Compositions and methods of use of antibacterial drug combinations

ABSTRACT

The present disclosure encompasses antibacterial compositions and methods of treating bacterial infections caused by resistant bacteria.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. patent application Ser. No.15/743,203, filed Jan. 9, 2018, which claims the benefit of PCTApplication number PCT/US/2016/041565, filed Jul. 8, 2016, which claimsthe benefit of U.S. Provisional Application No. 62/190,588, filed Jul.9, 2015, the disclosure disclosures of which is are hereby incorporatedby reference in its theft entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under DK098089,GM099538, A1090818, A1104987, GM007067, and HG000045 awarded by theNational Institutes of Health and DGE1143954 awarded by the NationalScience Foundation. The government has certain rights in the invention.

FIELD OF THE INVENTION

The disclosure relates to antibacterial compositions and methods oftreating bacterial infections caused by resistant bacteria.

BACKGROUND OF THE INVENTION

Multidrug-resistant (MDR) pathogens represent a growing threat to humanhealth, with many infectious diseases effectively regressing toward thepre-antibiotic era, exemplified by the dramatic rise ofcommunity-acquired methicillin-resistant Staphylococcus aureus (MRSA)infections. Methicillin-resistant Staphylococcus aureus (MRSA) is one ofthe most prevalent multidrug-resistant pathogens worldwide, exhibitingincreasing resistance to the latest antibiotic monotherapies used totreat these infections. The emergence of MRSA has virtually eliminatedthe use of β-lactams as therapeutic options against S. aureus. Therecently developed β-lactam agent ceftaroline, which exhibits activityin treatment of MRSA infections, does so by binding to the allostericsite of PBP2a, triggering opening of the active site for inactivation bythe drug; however, resistance to ceftaroline and other antibiotics usedto treat MRSA, including linezolid, vancomycin, and daptomycin, has beenreported. Thus, there is a need in the art for a new strategy to treatMDR pathogens and repurpose existing antibiotics.

SUMMARY OF THE INVENTION

In an aspect, the disclosure provides a composition useful for thetreatment of an infection caused be an antibiotic resistant bacterium,wherein the resistance is due to a penicillin-binding protein 2a(PBP2a)-driven mechanism. The composition comprises: (i) at least onecarbapenem or other suitable β-lactam capable of binding the allostericsite of PBP2a; (ii) at least one β-lactamase inhibitor; and (iii) atleast one β-lactam that binds the open configuration of the active siteof PBP2a. In certain embodiments, the antibiotic resistant bacterium ismethicillin-resistant Staphylococcus aureus (MRSA). In one embodiment,the carbapenem is meropenem, the β-lactamase inhibitor is tazobactam andthe β-lactam that binds the open configuration of the active site ofPBP2a is piperacillin. In another embodiment, the carbapenem isimipenem, the β-lactamase inhibitor is clavulanate and the β-lactam thatbinds the open configuration of the active site of PBP2a ispiperacillin. In still another embodiment, the carbapenem is meropenem,the β-lactamase inhibitor is tazobactam and the β-lactam that binds theopen configuration of the active site of PBP2a is amoxicillin.Specifically, the composition suppresses the evolution of resistance tothe composition.

In another aspect, the disclosure provides a method for treating aninfection caused by an antibiotic resistant bacterium in a subject,wherein the resistance is due to a penicillin-binding protein 2a(PBP2a)-driven mechanism. The method comprises administering to thesubject an effective amount of a composition of the disclosure.

In still another aspect, the disclosure provides a method for treatingan infection caused by methicillin-resistant Staphylococcus aureus(MRSA). The method comprises administering to the subject an effectiveamount of a composition of the disclosure.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1 depicts a 3D-checkerboard synergy determination showing isobolesof minimal inhibitory concentrations (MIC) and in vitro grown insingle-, double-, or triple-drug conditions of ME/PI/TZ. Coloredlines/isoboles within each panel indicate MICs of two drugs incombination. Dashed lines indicate theoretical concentrations ofadditive interactions. Points indicate top sub-inhibitory concentrationsof meropenem (ME), piperacillin (PI) and tazobactam (TZ) for each testedcondition. The red triangle indicates the MIC of all three drugs incombination (Each at 2 μg/ml).

FIG. 2 depicts a graph displaying the change in growth rates over timeof MRSA N315 when challenged with antibacterial combinations. Growthrates of MRSA N315 over an 11-day period were computed for eachantibacterial combination tested at one threefold dilution below MIC.The differences in growth rate (Δr) between day one (Initial GrowthRate) and the averaged rate of the last six days of the assay (FinalGrowth Rate) were calculated. MRSA N315 in conditions whose change ingrowth rate Δr>0.2 were considered to be adapted. The adaptation timeparameter t-adapt was calculated as the time at which change in growthrate was half-maximal. Adaptation rate, α=(Δr/2)/t-adapt (1/h²), wascomputed for strains meeting this criterion. Results are from tworeplicate experiments. rate for ME/PI: α=8.23×10⁻³ h⁻²; ME/TZ:α=8.68×10⁻⁴ h⁻²; PI/TZ: α=4.32×10⁻³ h⁻². Only ME/PI/TZ at one threefolddilution below MIC (1.2 μg/ml each) and No-drug Control displayed lackof increase in growth rate and were non-adapted.

FIG. 3A and FIG. 3B depict diagrams illustrating the collateralsensitivities that underlie suppression of adaptation to antibacterialcombinations in MRSA N315. (FIG. 3A) MRSA N315 interaction network ofcollateral sensitivities and resistance between ME/PI/TZ, its single anddouble constituents, and other 13-lactam compounds of varioussub-classes (cephalosporins, penicillins, carbapenems, and 13-lactamaseinhibitors). Node colors indicate sub-classes of 13-lactams,13-lactamase inhibitors, or combinations. Blue arrows indicatecollateral sensitivities. Black lines indicate collateral resistance.For example, adaptation to piperacillin sensitizes MRSA N315 tomeropenem and imipenem. Cephalosporins were not collaterally sensitiveto any of the compounds we tested. Where pairs were not tested or nocollateral effects were seen, no connecting arrows are shown. (FIG. 3B)MRSA N315 interaction network of collateral sensitivities and resistancebetween ME/PI/TZ and its single and double constituents only. Bold bluearrows indicate reciprocal collateral sensitivities between two nodes,e.g., piperacillin and meropenem/tazobactam.

FIG. 4A, FIG. 4B and FIG. 4C depict graphs showing genomic evidence formechanisms of synergy and collateral sensitivity. (FIG. 4A) Adaptationof MRSA N315 to meropenem/tazobactam or tazobactam alone destabilizesplasmid pN315. Read coverage aligning to pN315 in MRSA N315 adapted todrug combinations containing tazobactam (TZ) or not containingtazobactam (non-TZ), versus total reads per sample. Days of adaptationunder the given conditions are indicated, e.g., D-2 indicates isolatewas sequenced after two days of adaptation. (FIG. 4B, FIG. 4C) qRT-PCRconfirms disregulation of the bla and mec operons as causativemechanisms of some collateral sensitivities in MRSA N315. Expression ofblaZ or mecA shown relative to gyrB in wild-type MRSA N315 or adaptedstrains (N315 adapted to TZ 100 μg/ml, and PI 33.3 or 100 μg/ml),subsequently grown in broth-only or broth+sub-MIC PI or TZ. N.D.=Notdetermined. “−” indicates no expression. Loss of blaZ expression in MRSAN315 adapted to TZ confirms loss of blaZ and the bla operon, and isconsistent with disregulation of mecA expression. Data are from threereplicate experiments. Error bars indicate standard error ofmeasurement.

FIG. 5 depicts a graph showing efficacy of ME/PIT/TZ treatment inneutropenic mouse peritonitis model of MRSA N315. Proportional survivalof mice (n=6) from each drug treatment is shown. Treatment withME/PI/TZ, ME/PI, and linezolid are significantly different than vehicle(*p=0.02). Error bars indicate standard error of proportions ofsurvivors per condition tested.

FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6Hand FIG. 6I depict bacterial plate growth illustrating PBP xyloseinduction for MRSA COL antisense (AS) strains. (FIG. 6A, FIG. 6B, FIG.6C) pbp2a/mecA antisense (AS) strain. Targeted repression of PBP2ashowed increased susceptibility for meropenem when under xyloseinduction. Increased susceptibility was also observed for piperacillin.Increased susceptibility was observed for all combinations. (FIG. 6D,FIG. 6E) pbpA antisense (AS) strain. Targeted repression of PBP1 showedincreased susceptibility to meropenem and piperacillin. Increasedsusceptibility was observed for the ME/PI, ME/TZ, and ME/PI/TZcombinations. (FIG. 6F, FIG. 6G) pbp2 antisense (AS) strain. Targetedrepression of PBP2 showed increased susceptibility to meropenem andpiperacillin. Increased susceptibility was observed for allcombinations. (FIG. 6H, FIG. 6I) pbp3 antisense (AS) strain. Targetedrepression of PBP3 showed no increase in susceptibility to any of thesingle drugs. A slight increase in susceptibility was observed for theME/PI combination; no change in susceptibility was observed for any ofthe other combinations.

FIG. 7 depicts a plot illustrating collateral sensitivities underlyingthe suppression of adaptation to β-lactam combinations in MRSA N315.Blue shades indicate collateral sensitization of strain to single drugsand combinations, after prior adaptation to single and double drugcombinations. Red shades indicate collateral resistance. Lightshading=change of one MIC dilution. Dark shading=change of two or moreMIC dilutions. For example, adaptation to piperacillin yields collateralsensitivity to meropenem, and vice versa. ME=meropenem, PI=piperacillin,TZ=tazobactam, AX=amoxicillin, CF=cefdinir, CP=cefepime, CX=cefoxitin,DC=dicloxacillin, IM=imipenem.

FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D depict histograms illustrating thegenomic duplication in MRSA N315 adapted to piperacillin/tazobactam.Histogram showing the total read coverage across the genome of N315adapted to FIG. 8A, meropenem/tazobactam for five days, FIG. 8B,tazobactam alone for two days, FIG. 8C, piperacillin/tazobactam for sixdays, and FIG. 8D, piperacillin/tazobactam for 11 days. Average per-baseread coverage across the entire genome and only in the region indicatedby the red box are, respectively: a) 116.6 reads/bp and 126.6 reads/bp;b) 124.5 reads/bp and 128.9 reads/bp; c) 157.8 reads/bp and 302.2reads/bp; and d) 120.1 reads/bp and 230.6 reads/bp. Clones in FIG. 8Aand FIG. 8B were chosen to be representative of allnon-piperacillin/tazobactam adaptations.

FIG. 9 depicts a proposed mechanism of synergy ofmeropenem/piperacillin/tazobactam (ME/PI/TZ) against MRSA. Our datasupport the proposed synergistic mode of action against cell-wallsynthesis in MRSA involving: I.) suppression of transpeptidation by PBP1at the division septum by carbapenems, II.) suppression oftranspeptidation by PBP2 by penams (penicillins), Ill.) suppression ofβ-lactamase activity against penams by β-lactamase inhibitors, and IV.)allosteric opening of the active site of PBP2a by meropenem, allowinginhibition by meropenem or by other β-lactams.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are compositions comprising a combination ofantibiotics from three subclasses of β-lactam antibiotics, all targetingcell-wall synthesis. This therapy uses: 1) antibiotics that targetmultiple nodes in the same cellular system, 2) combinations of theseantibiotics that increase drug potency by utilizing drug synergy, and 3)collateral sensitivity between constituents of the combination tosuppress resistance evolution. Without wishing to be bound by theory,the composition comprises a carbapenem or other suitable β-lactam whichinhibits penicillin-binding protein 1 (PBP1) and allosterically opensthe active site of PBP2a for inhibition by another molecule ofantibiotic in the combination, a β-lactam that binds PBP2 and PBP2a, anda β-lactamase inhibitor the protects the β-lactam(s) from β-lactamases.This culminates in a synergistic response by simultaneous perturbationof multiple components of the cell-wall synthesis machinery. Theinventors have shown that the combination acts synergistically and isbactericidal against bacteria comprising PBP2a, specificallymethicillin-resistant Staphylococcus aureus (MRSA).

I. Compositions

In an aspect, provided herein are composition useful for the treatmentof an infection caused be an antibiotic resistant bacterium, wherein theresistance is due to a penicillin-binding protein 2a (PBP2a)-drivenmechanism. The composition comprises at least one carbapenem or othersuitable β-lactams capable of binding the allosteric site of PBP2a; atleast one β-lactamase inhibitor; and at least one β-lactam that bindsthe open configuration of the active site of PBP2a.

The inventors have discovered that the disclosed composition reduces oreliminates the evolution of resistant bacteria following exposure to therecited combination of antibiotics. In the disclosed composition, it isessential that one antibiotic bind the allosteric site of PBP2a and thatanother antibiotic binds the active site of PBP2a. Without wishing to bebound by theory, the antibiotic that binds the allosteric site of PBP2aopens the transpeptidase active site of PBP2a such that the antibioticthat binds the active site of PBP2a may also bind to a separate site onPBP2a. Binding of both these antibiotics is required to completely stopthe vital transpeptidase function of PBP2a. By stopping thetranspeptidase function of PBP2a, an antibiotic resistant bacterium thatrelies on a PBP2a-driven mechanism for resistance will be killed. Onespecific example of such a bacterium is methicillin-resistantStaphylococcus aureus (MRSA). The β-lactamase inhibitor of thecomposition further facilitates killing of the resistant bacteria byprotecting the β-lactam(s) in the combination from enzymatic breakdownby β-lactamases produced by the resistant bacteria.

According to the disclosure, the composition is effective againstantibiotic-resistant bacteria, wherein the resistance is due to apenicillin-binding protein 2a (PBP2a)-driven mechanism. A skilledartisan would be able to determine if the antibiotic-resistant bacteriaexhibits resistance due to a PBP2a-driven mechanism. For example, thebacteria may comprise mecA, which encodes for PBP2a. Generally, specieswithin the genus Staphylococcus are known to possess the mecA gene atthis time, including: S. aureus, S. epidermidis, S. hominis, S.lugdunensis, S. xylosus, and S. fells (Petinaki et al. Detection ofmecA, mecR1 and mecI genes among clinical isolates ofmethicillin-resistant staphylococci by combined polymerase chainreactions. J. Antimicrob. Chemother. 47, 297-304 (2001)), (Nascimento etal. Potential spread of multidrug-resistant coagulase-negativestaphylococci through healthcare waste. J. Infect. Dev. Ctries. 9,(2015)). Accordingly, in an embodiment, an antibiotic-resistantbacterium is from the genus Staphylococcus. In another embodiment, anantibiotic-resistant bacterium is selected from the group consisting ofS. aureus, S. epidermidis, S. hominis, S. lugdunensis, S. xylosus, andS. fells. In a specific embodiment, the antibiotic-resistant bacteriumis methicillin-resistant Staphylococcus aureus (MRSA). PBPs arepenicillin-binding proteins, which are the transpeptidase enzymesnecessary for synthesis of the peptidoglycan cell wall in bacteria.There are four native PBPs in Staphylococcus aureus, of which PBP2 isthe essential PBP for viability of the cell. In addition to possessingPBP2, methicillin-resistant S. aureus (MRSA) has acquired an accessoryPBP (PBP2a) that is able to perform all critical transpeptidase activityfor cell wall synthesis. PBP2a has transpeptidase activity even in thepresence of many β-lactams which typically function by inhibiting thenative PBP2. The β-lactam drugs specifically target PBPs, and arecomposed of several subclasses which all contain the β-lactam “warhead”,have varying PBP affinities. Non-limiting examples include thepenicillins, carbapenems, cephalosporins, and monobactams. However,PBP2a has poor binding affinity with some of the β-lactams while it isin its native, closed conformation, resulting in high resistance to thisdrug class by PBP2a-containing organisms. Accordingly, the presentdisclosure overcomes this poor binding by employing one β-lactam thatbinds to the allosteric site of PBP2a thus keeping it open while asecond β-lactam binds to the open configuration of the active sitethereby effectively inhibiting the function of PBP2a.

A composition of the disclosure comprises, in part, a carbapenem orother suitable β-lactams capable of binding the allosteric site ofPBP2a. Accordingly, a carbapenem or suitable β-lactam of the disclosuremust bind to the allosteric site of PBP2a. Non-limiting examples ofcarbapenems or β-lactams that bind to the allosteric site of PBP2ainclude meropenem, imipenem, tomopenem, ceftaroline and ceftobiprole. Ina specific embodiment, the carbapenem is selected from the groupconsisting of meropenem and imipenem. In another specific embodiment,the carbapenem is meropenem. In still another specific embodiment, thecarbapenem is imipenem.

A composition of the disclosure comprises, in part, a β-lactamaseinhibitor. A suitable β-lactamase inhibitor protects the β-lactam alsopresent in the composition from enzymatic breakdown by β-lactamasesproduced by the bacteria. Accordingly, a β-lactamase inhibitor inhibitsthe activity of β-lactamases, a family of enzymes that break theβ-lactam ring that allows penicillin-like antibiotics to work.Non-limiting examples of β-lactamase inhibitors include clavulanic acid(clavulanate), sulbactam, tazobactam, and avibactam. In a specificembodiment, the β-lactamase inhibitor is selected from the groupconsisting of tazobactam and clavulanate. In another specificembodiment, the β-lactamase inhibitor is tazobactam. In still anotherspecific embodiment, the β-lactamase inhibitor is clavulanate.

A composition of the disclosure comprises, in part, a β-lactam thatbinds the open configuration of the active site of PBP2a. Accordingly, aβ-lactam of the disclosure must bind to the open configuration of theactive site of PBP2a. Non-limiting examples of β-lactams that bind tothe open configuration of the active site of PBP2a include carbapenems,aminopenicillins, carboxypenicillins, ureidopenicillins, oxacillins,methicillins, and some cephalosporins. Non-limiting examples ofcarbapenems include meropenem, imipenem, doripenem, ertapenem,faropenem, and tebipenem. Non-limiting examples of oxacillins andmethicillins include cloxacillin, dicloxacillin, flucloxacillin,oxacillin, methicillin and nafcillin. Non-limiting examples ofcephalosporins that bind to the open configuration of the active site ofPBP2a include cefepime, cefozopran, cefpirome, cefquinome, ceftaroline,and ceftobiprole. Notably, most penicillin-type β-lactams (e.g.aminopenicillins, carboxypenicillins, ureidopenicillins) bind to theopen configuration of the active site of PBP2a. Non-limiting examples ofaminopenicillins include amoxicillin, ampicillin, pivampicillin,hetacillin, bacampicillin, metampicillin, talampicillin, and epicillin.Non-limiting examples of carboxypenicillins include carbenicillin,carindacillin, ticarcillin and temocillin. Non-limiting examples ofureidopenicillins include azlocillin, mezlocillin and piperacillin.Specific non-limiting examples of β-lactams that do not bind to the openconfiguration of the active site of PBP2a and will not work in acomposition of the disclosure include mecillinam, cefradine andthienamycin. In a specific embodiment, the β-lactam that binds the openconfiguration of the active site of PBP2a is selected from the groupconsisting of piperacillin and amoxicillin. In another specificembodiment, the β-lactam that binds the open configuration of the activesite of PBP2a is piperacillin. In still another specific embodiment, theβ-lactam that binds the open configuration of the active site of PBP2ais amoxicillin.

In a specific embodiment, the carbapenem or other suitable β-lactamcapable of binding the allosteric site of PBP2a is meropenem, theβ-lactamase inhibitor is tazobactam and the β-lactam that binds the openconfiguration of the active site of PBP2a is piperacillin. In anotherspecific embodiment, the carbapenem or other suitable β-lactam capableof binding the allosteric site of PBP2a is imipenem, the β-lactamaseinhibitor is clavulanate and the β-lactam that binds the openconfiguration of the active site of PBP2a is piperacillin. In stillanother specific embodiment, the carbapenem or other suitable β-lactamcapable of binding the allosteric site of PBP2a is meropenem, theβ-lactamase inhibitor is tazobactam and the β-lactam that binds the openconfiguration of the active site of PBP2a is amoxicillin.

The ratio of the amount of each antibiotic in the combination may beexperimentally determined via methods known in the art. For example, a3-D checkerboard graph may be used to determine the ratio of eachantibiotic in the combination as depicted in FIG. 1. Using such amethodology, a skilled artisan may be able to determine the optimalratio of the antibiotic combination. As demonstrated by the inventors,the ratio of the antibiotics in the combination may range from 64:1:1,1:64:1, and 1:1:64 to 1:1:1. In a specific embodiment, the ratio ofcarbapenem or other suitable β-lactam capable of binding the allostericsite of PBP2a to β-lactamase inhibitor to β-lactam that binds the openconfiguration of the active site of PBP2a is 1:1:1. However, it isunderstood that these ratios may be varied and still result in aneffective combination of the disclosure.

Advantageously, a composition of the disclosure suppresses the evolutionof resistance to said composition. Often patients treated with aspecific antibiotic development resistance to said antibiotic due tovarious reasons. The development and spread of resistance candramatically dampen the effectiveness and longevity of antimicrobialtherapy. Surprisingly, the inventors have shown that a combination ofthe disclosure suppresses the evolution of resistance. Stated anotherway, after prolonged exposure of the bacteria to a combination of thedisclosure, no bacteria resistant to said combination were observed.This is in stark contrast to both single drug and dual drugcombinations.

(a) Pharmaceutical Composition

The present disclosure also provides pharmaceutical compositions. Thepharmaceutical composition comprises one or more antibiotics of thedisclosure, as an active ingredient, and at least one pharmaceuticallyacceptable excipient.

The pharmaceutically acceptable excipient may be a diluent, a binder, afiller, a buffering agent, a pH-modifying agent, a disintegrant, adispersant, a preservative, a lubricant, taste-masking agent, aflavoring agent, or a coloring agent. The amount and types of excipientsutilized to form pharmaceutical compositions may be selected accordingto known principles of pharmaceutical science.

In one embodiment, the excipient may be a diluent. The diluent may becompressible (i.e., plastically deformable) or abrasively brittle.Non-limiting examples of suitable compressible diluents includemicrocrystalline cellulose (MCC), cellulose derivatives, cellulosepowder, cellulose esters (i.e., acetate and butyrate mixed esters),ethyl cellulose, methyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, sodium carboxymethylcellulose, cornstarch, phosphated corn starch, pregelatinized corn starch, rice starch,potato starch, tapioca starch, starch-lactose, starch-calcium carbonate,sodium starch glycolate, glucose, fructose, lactose, lactosemonohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol,xylitol, maltodextrin, and trehalose. Non-limiting examples of suitableabrasively brittle diluents include dibasic calcium phosphate (anhydrousor dihydrate), calcium phosphate tribasic, calcium carbonate, andmagnesium carbonate.

In another embodiment, the excipient may be a binder. Suitable bindersinclude, but are not limited to, starches, pregelatinized starches,gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodiumcarboxymethylcellulose, ethylcellulose, polyacrylamides,polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fatty acid alcohol,polyethylene glycol, polyols, saccharides, oligosaccharides,polypeptides, oligopeptides, and combinations thereof.

In another embodiment, the excipient may be a filler. Suitable fillersinclude, but are not limited to, carbohydrates, inorganic compounds, andpolyvinylpyrrolidone. By way of non-limiting example, the filler may becalcium sulfate, both di- and tri-basic, starch, calcium carbonate,magnesium carbonate, microcrystalline cellulose, dibasic calciumphosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc,modified starches, lactose, sucrose, mannitol, or sorbitol.

In still another embodiment, the excipient may be a buffering agent.Representative examples of suitable buffering agents include, but arenot limited to, phosphates, carbonates, citrates, tris buffers, andbuffered saline salts (e.g., Tris buffered saline or phosphate bufferedsaline).

In various embodiments, the excipient may be a pH modifier. By way ofnon-limiting example, the pH modifying agent may be sodium carbonate,sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

In a further embodiment, the excipient may be a disintegrant. Thedisintegrant may be non-effervescent or effervescent. Suitable examplesof non-effervescent disintegrants include, but are not limited to,starches such as corn starch, potato starch, pregelatinized and modifiedstarches thereof, sweeteners, clays, such as bentonite,micro-crystalline cellulose, alginates, sodium starch glycolate, gumssuch as agar, guar, locust bean, karaya, pecitin, and tragacanth.Non-limiting examples of suitable effervescent disintegrants includesodium bicarbonate in combination with citric acid and sodiumbicarbonate in combination with tartaric acid.

In yet another embodiment, the excipient may be a dispersant ordispersing enhancing agent. Suitable dispersants may include, but arenot limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum,kaolin, bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose.

In another alternate embodiment, the excipient may be a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate,citric acid, sodium citrate; chelators such as EDTA or EGTA; andantimicrobials, such as parabens, chlorobutanol, or phenol.

In a further embodiment, the excipient may be a lubricant. Non-limitingexamples of suitable lubricants include minerals such as talc or silica;and fats such as vegetable stearin, magnesium stearate or stearic acid.

In yet another embodiment, the excipient may be a taste-masking agent.Taste-masking materials include cellulose ethers; polyethylene glycols;polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers;monoglycerides or triglycerides; acrylic polymers; mixtures of acrylicpolymers with cellulose ethers; cellulose acetate phthalate; andcombinations thereof.

In an alternate embodiment, the excipient may be a flavoring agent.Flavoring agents may be chosen from synthetic flavor oils and flavoringaromatics and/or natural oils, extracts from plants, leaves, flowers,fruits, and combinations thereof.

In still a further embodiment, the excipient may be a coloring agent.Suitable color additives include, but are not limited to, food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drugand cosmetic colors (Ext. D&C).

The weight fraction of the excipient or combination of excipients in thecomposition may be about 99% or less, about 97% or less, about 95% orless, about 90% or less, about 85% or less, about 80% or less, about 75%or less, about 70% or less, about 65% or less, about 60% or less, about55% or less, about 50% or less, about 45% or less, about 40% or less,about 35% or less, about 30% or less, about 25% or less, about 20% orless, about 15% or less, about 10% or less, about 5% or less, about 2%,or about 1% or less of the total weight of the composition.

The composition can be formulated into various dosage forms andadministered by a number of different means that will deliver atherapeutically effective amount of the active ingredient. Suchcompositions can be administered orally, parenterally, or topically indosage unit formulations containing conventional nontoxicpharmaceutically acceptable carriers, adjuvants, and vehicles asdesired. Topical administration may also involve the use of transdermaladministration such as transdermal patches or iontophoresis devices. Theterm parenteral as used herein includes subcutaneous, intravenous,intramuscular, or intrasternal injection, or infusion techniques.Formulation of drugs is discussed in, for example, Gennaro, A. R.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.(18^(th) ed, 1995), and Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980).

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, powders, pellets, and granules. In such solid dosageforms, the active ingredient is ordinarily combined with one or morepharmaceutically acceptable excipients, examples of which are detailedabove. Oral preparations may also be administered as aqueoussuspensions, elixirs, or syrups. For these, the active ingredient may becombined with various sweetening or flavoring agents, coloring agents,and, if so desired, emulsifying and/or suspending agents, as well asdiluents such as water, ethanol, glycerin, and combinations thereof.

For parenteral administration (including subcutaneous, intradermal,intravenous, intramuscular, and intraperitoneal), the preparation may bean aqueous or an oil-based solution. Aqueous solutions may include asterile diluent such as water, saline solution, a pharmaceuticallyacceptable polyol such as glycerol, propylene glycol, or other syntheticsolvents; an antibacterial and/or antifungal agent such as benzylalcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and thelike; an antioxidant such as ascorbic acid or sodium bisulfite; achelating agent such as etheylenediaminetetraacetic acid; a buffer suchas acetate, citrate, or phosphate; and/or an agent for the adjustment oftonicity such as sodium chloride, dextrose, or a polyalcohol such asmannitol or sorbitol. The pH of the aqueous solution may be adjustedwith acids or bases such as hydrochloric acid or sodium hydroxide.Oil-based solutions or suspensions may further comprise sesame, peanut,olive oil, or mineral oil.

For topical (e.g., transdermal or transmucosal) administration,penetrants appropriate to the barrier to be permeated are generallyincluded in the preparation. Transmucosal administration may beaccomplished through the use of nasal sprays, aerosol sprays, tablets,or suppositories, and transdermal administration may be via ointments,salves, gels, patches, or creams as generally known in the art.

II. Methods

In an aspect, the present disclosure encompasses a method for treatingan infection caused by an antibiotic resistant bacterium in a subject,wherein the resistance is due to a penicillin-binding protein 2a(PBP2a)-driven mechanism. The method comprises administering to thesubject an effective amount of a composition of the disclosure.

In another aspect, the present disclosure encompasses a method fortreating an infection caused by methicillin-resistant Staphylococcusaureus (MRSA). The method comprises administering to the subject aneffective amount of a composition of the disclosure.

The term “infection” as used herein includes presence of microbes,including bacteria, in or on a subject, which, if its growth wereinhibited, would result in a benefit to the subject. As such, the term“infection” in addition to referring to the presence of bacteria alsorefers to normal flora which, are not desirable. The term “infection”includes infection caused by bacteria.

The term “treat”, “treating” or “treatment” as used herein refers toadministering a pharmaceutical composition of the disclosure forprophylactic and/or therapeutic purposes. The term “prophylactictreatment” refers to treating a subject who is not yet infected, but whois susceptible to, or otherwise at a risk of infection. The term“therapeutic treatment” refers to administering treatment to a subjectalready suffering from infection. The term “treat”, “treating” or“treatment” as used herein also refers to administering a composition ofthe disclosure, with or without additional pharmaceutically active orinert ingredients, in order to: (i) reduce or eliminate either abacterial infection or one or more symptoms of the bacterial infection,or (ii) retard the progression of a bacterial infection or of one ormore symptoms of the bacterial infection, or (iii) reduce the severityof a bacterial infection or of one or more symptoms of the bacterialinfections, or (iv) suppress the clinical manifestation of a bacterialinfection, or (v) suppress the manifestation of adverse symptoms of thebacterial infections.

The term “control” or “controlling” as used herein generally refers topreventing, reducing, or eradicating infection or inhibiting the rateand extent of such infection, or reducing the microbial population, suchas a microbial population present in or on a body or structure, surface,liquid, subject, etc, wherein such prevention or reduction in theinfection or microbial population is statistically significant withrespect to untreated infection or population. In general, such controlmay be achieved by increased mortality amongst the microbial population.

The term “effective amount” as used herein refers to an amount, whichhas a therapeutic effect or is the amount required to produce atherapeutic effect in a subject. For example, a therapeutically orpharmaceutically effective amount of an antibiotic agent or apharmaceutical composition is the amount of the antibiotic agent or thepharmaceutical composition required to produce a desired therapeuticeffect as may be judged by clinical trial results, model animalinfection studies, and/or in vitro studies (e.g. in agar or brothmedia). The effective or pharmaceutically effective amount depends onseveral factors, including but not limited to, the microorganism (e.g.bacteria) involved, characteristics of the subject (for example height,weight, sex, age and medical history), severity of infection and theparticular type of the antibiotic used. For prophylactic treatments, atherapeutically or prophylactically effective amount is that amountwhich would be effective to prevent a microbial (e.g. bacterial)infection. Notably, the combination disclosed herein may allow foreffective amount of the combination of active agents to be lower thanthe effective amount alone such that a lower total concentration ofantibiotic is administered to a subject.

The term “administration” or “administering” includes delivery of acomposition or one or more pharmaceutically active ingredients to asubject, including for example, by any appropriate methods, which servesto deliver the composition or it's active ingredients or otherpharmaceutically active ingredients to the site of the infection. Themethod of administration can vary depending on various factors, such asfor example, the components of the pharmaceutical composition or thetype/nature of the pharmaceutically active or inert ingredients, thesite of the potential or actual infection, the microorganism involved,severity of the infection, age and physical condition of the subject.Some non-limiting examples of ways to administer a composition or apharmaceutically active ingredient to a subject according to thisdisclosure includes oral, intravenous, topical, intrarespiratory,intraperitoneal, intramuscular, parenteral, sublingual, transdermal,intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal,gene gun, dermal patch, eye drop, ear drop or mouthwash. In someembodiments, the compositions or active ingredients thereof areadministered orally.

The compositions according to the disclosure can be formulated intovarious dosage forms wherein the active ingredients may be presenteither together (e.g. as an admixture) or as separate components. Whenthe various ingredients in the composition are formulated as a mixture,such composition can be delivered by administering such a mixture (e.g.in the form of a suitable unit dosage form such as tablet, capsule,solution, powder etc.). Alternatively, the ingredients may also beadministered separately (simultaneously or one after the other) as longas these ingredients reach beneficial therapeutic levels such that thecomposition as a whole provides a synergistic effect. The composition ordosage form wherein the ingredients do not come as a mixture, but comeas separate components, such composition/dosage form can be administeredin several ways. In one possible way, the ingredients can be mixed inthe desired proportions and the mixture is then administered asrequired. Alternatively, the components can be separately administeredin appropriate proportions so as to achieve the same or equivalenttherapeutic level or effect as would have been achieved byadministration of the equivalent mixture.

A subject may be a human, a livestock animal, a companion animal, a labanimal, or a zoological animal. In one embodiment, the subject may be arodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment,the subject may be a livestock animal. Non-limiting examples of suitablelivestock animals may include pigs, cows, horses, goats, sheep, llamasand alpacas. In yet another embodiment, the subject may be a companionanimal. Non-limiting examples of companion animals may include pets suchas dogs, cats, rabbits, and birds. In yet another embodiment, thesubject may be a zoological animal. As used herein, a “zoologicalanimal” refers to an animal that may be found in a zoo. Such animals mayinclude non-human primates, large cats, wolves, and bears. In certainembodiments, the animal is a laboratory animal. Non-limiting examples ofa laboratory animal may include rodents, canines, felines, and non-humanprimates. In certain embodiments, the animal is a rodent. Non-limitingexamples of rodents may include mice, rats, guinea pigs, etc.

In general, the pharmaceutical compositions and method disclosed hereinare useful in treating or controlling bacterial infections caused byantibiotic resistant bacteria, wherein the resistance is due to apenicillin-binding protein 2a (PBP2a)-driven mechanism. Somenon-limiting examples of such bacteria known to have developedresistance due to PBP2a include S. aureus, S. epidermidis, S. hominis,S. lugdunensis, S. xylosus, and S. fells (Petinaki et al. Detection ofmecA, mecR1 and mecl genes among clinical isolates ofmethicillin-resistant staphylococci by combined polymerase chainreactions. J. Antimicrob. Chemother. 47, 297-304 (2001)), (Nascimento etal. Potential spread of multidrug-resistant coagulase-negativestaphylococci through healthcare waste. J. Infect. Dev. Ctries. 9,(2015)). In a specific embodiment, the antibiotic resistant bacterium ismethicillin-resistant Staphylococcus aureus (MRSA). Non-limitingexamples of infections that may be prevented or treated using thecompositions and/or methods of the disclosure include: skin and softtissue infections, febrile neutropenia, urinary tract infection,intraabdominal infections, respiratory tract infections, pneumonia(nosocomial), bacteremia meningitis, and surgical infections.

EXAMPLES

The following examples are included to demonstrate various embodimentsof the present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Introduction to the Examples

Multidrug-resistant (MDR) pathogens represent a growing threat to humanhealth, with many infectious diseases effectively regressing toward thepre-antibiotic era¹⁻³, exemplified by the dramatic rise ofcommunity-acquired methicillin-resistant Staphylococcus aureus (MRSA)infections. In the 1940's, S. aureus infections were primarily treatedwith first-generation β-lactams (penicillins), which target thepenicillin-binding proteins (PBPs), the critical transpeptidases forcell-wall synthesis⁴. Four PBPs (PBP1-PBP4) perform these functions inS. aureus ⁴. Emergence of β-lactamase-producing strains led todevelopment of β-lactamase-resistant second-generation penicillins,including methicillin. Soon after the introduction of methicillin in1959, the first MRSA strains were reported⁵. These strains acquired ahighly regulated collection of genes from a non-S. aureus source thatproduced inducible resistance to β-lactam antibiotics⁴. One of thesegenes, mecA, encodes penicillin-binding protein 2a (PBP2a). PBP2aperforms the critical transpeptidase reaction that cross-links the cellwall, even under challenge by β-lactam antibiotics, when other PBPs areinhibited⁶⁻⁸. The mechanistic basis for this outcome is complex,involving a closed conformation for the active site, whose function isregulated by allostery^(9,10) The emergence of MRSA has virtuallyeliminated the use of β-lactams as therapeutic options against S.aureus. The recently developed β-lactam agent ceftaroline, whichexhibits activity in treatment of MRSA infections, does so by binding tothe allosteric site of PBP2a, triggering opening of the active site forinactivation by the drug^(10,11,) however, resistance to ceftaroline¹²and other antibiotics used to treat MRSA, including linezolid,vancomycin, and daptomycin, has been reported¹³⁻¹⁵.

Use of multidrug combination therapy targeting orthogonal cellularprocesses has been successful in treating Mycobacterium tuberculosis,Helicobacter pylori, and other infections^(16,17.) However, resistanceis increasing even against these therapies¹⁸⁻²⁰ We have identified a newpotential therapy against MRSA consisting of a combination of clinicallyapproved drugs from three distinct generations and subclasses ofβ-lactam antibiotics, all targeting cell-wall synthesis: meropenem,piperacillin, and tazobactam (ME/PI/TZ). This therapy uses elements fromthree strategies: 1) use of semi-synthetic antibiotic derivatives thattarget multiple nodes in the same cellular system^(12,21,)2) use ofcombinations of these antibiotics that increase drug potency byutilizing drug synergy^(22,23), and 3) use of collateral sensitivitybetween constituents of the combination to suppress resistanceevolution^(24,25.) Each of these methods have been successfully employedagainst the major MDR Gram-negative and Gram-positive humanpathogens^(26,27). However, these strategies used individually haveoften been thwarted by the evolution of new resistance in MDR pathogens,leading to diminishing options for treating theirinfections^(5,14,21,28,29).

We hypothesize that ME/PI/TZ operates through inhibition of PBP1 bymeropenem, the targeting of PBP2 by piperacillin, protection ofpiperacillin from the PC1 class A β-lactamases by tazobactam^(6,30-34,)and allosteric opening of the active site of PBP2a by meropenem forinhibition by another molecule of antibiotic in the combination¹¹. Thisculminates in a synergistic response by simultaneous perturbation ofmultiple components of the cell-wall synthesis machinery in MRSA. Wefind that exposure of MRSA N315 to the components of ME/PI/TZ revealsreciprocal collateral sensitivities within this highly synergistictriple combination that suppress the evolution of resistance, incontrast to some synergistic combination therapies that insteadaccelerate resistance evolution^(23,35.) This effect is consistent withrecent work showing that collateral sensitivity slows evolution ofresistance in a non-pathogenic laboratory strain of Escherichia coli^(24,36.) Our results support renewed clinical use of older β-lactamantibiotics against MRSA when used in synergistic combinations ofcollaterally sensitive components, opening a new treatment paradigm withexisting drugs that are already approved for human use.

Example 1. Synergy Between Meropenem, Piperacillin, and Tazobactam inMRSA Strains In Vitro

Based on its high level of resistance against 23 diverse antibiotics(Table 1), S. aureus MRSA N315³⁷ was selected from a group of fullygenome-sequenced MDR strains of MRSA for this study. MRSA N315 containsthe staphylococcal chromosome cassette mec (SCCmec) type II encoding themec methicillin-resistance operon³⁸, as well as penicillinase plasmidpN315 containing the bla β-lactamase operon³⁹. From a focusedcombinatorial screen of these 23 antibiotic compounds, includingrepresentatives from every major drug class (Table 1), we identified thecombination of ME/PI/TZ to display highly synergistic, bactericidalactivity against MRSA N315 in vitro, using the metric of the fractionalinhibitory concentration index (FICI), FICI=0.11^(40,41) (Table 2A). Forany number of drugs in combination, a FICI less than 1 indicatessynergy, a FICI equal to 1 indicates additivity, and a FICI greater than1 indicates indifference or antagonism^(40,41). Notably, these threedrugs all belong to different sub-classes of the β-lactam drugs, whichtarget the critical transpeptidase enzymes of cell-wall synthesis,though MRSA strains are typically highly resistant to most β-lactams⁸.The general resistance to individual β-lactams results from theinability of these drugs to inhibit the transpeptidase active site ofPBP2a, which compensates for β-lactam inhibition of the othertranspeptidases in S. aureus ⁸.

ME/PI/TZ exhibits increased synergy against MRSA N315 relative to itsthree constituent double combinations meropenem/piperacillin (ME/PI),meropenem/tazobactam (ME/TZ) and piperacillin/tazobactam (PI/TZ) atclinically relevant concentrations (FIG. 1, Table 2B-C). All threeβ-lactam compounds were tested for final MIC and FICI using a 3-Dcheckerboard with twofold dilution series of each compound from 128-to-2μg/ml, and no-drug. These allowed up to a 64-fold difference incomponent ratios to be explored for maximal synergy, as well as allowingfor isolation of results for each single compound, all constituentdouble combinations, and the triple combination. Using the 3-Dcheckerboard, we determined the optimal ratio for ME/PI/TZ to be 1:1:1for minimal drug input and maximal synergy against MRSA N315. Theminimal-inhibitory concentrations (MICs) of the three components in thecombination against MRSA N315 (2 μg/ml each) are below the clinicalsusceptibility breakpoints for each of these drugs alone againstmethicillin-susceptible S. aureus (4-8 μg/ml)⁴². The constituent doublecombinations ME/PI and PI/TZ are also synergistic against N315 withFICI=0.44 and 0.22, respectively, while ME/TZ is less synergistic at0.67. Based on the Loewe additivity model of synergy, drugs cannot besynergistic with themselves³⁶. Though the β-lactams all target thecell-wall pathway, our use of the FICI method (Loewe additivity)confirms the non-additive nature of these interactions. In contrast tothe high synergy of ME/PI/TZ seen in MRSA N315, the combination exhibitsslightly less than additive activity (FICI=1.12) in themethicillin-susceptible S. aureus (MSSA) reference strain ATCC29213^(42,43) (Table 2B-C), and we hypothesize the necessity of PBP2afor synergy to occur.

We propose that the mechanism of synergy observed for ME/PI/TZ resultsfrom allosteric triggering of PBP2a by its constituents, akin to thatreported for ceftaroline^(10,11). Indeed, we determined that meropenembinds to the allosteric site of PBP2a with a dissociation constant(K_(d)) of 270±80 μM (equivalent to 104±31 μg/ml). The mean peak plasmaconcentration in healthy humans after a bolus intravenous injection ofmeropenem at the recommended 1 g dose is 112 μg/ml⁴⁴. The concentrationsof meropenem achieved clinically are above the K_(d); thus at theseconcentrations meropenem binding to the allosteric site of PBP2a wouldtrigger opening of the active site of PBP2a, enabling access to itstranspeptidase active site for acylation/inactivation either by anothermolecule of meropenem or by other β-lactams in thecombination^(8,10,45).

The highly synergistic activity of ME/PI/TZ against MRSA N315 wasrecapitulated against all in a panel of 72 clinical MRSA isolates withmultiple SCCmec types represented (Table 3A-B). The MIC of thecombination against the clinical isolates ranged from 0.4-33.3 μg/ml foreach component, with a mean of 9.7 μg/ml, and an MIC₅₀ and MIC₉₀ of 3.7μg/ml and 33.3 μg/ml, respectively (Table 4A).

Example 2. Mechanistic Robustness of Synergy Using AlternateCarbapenems, Penicillins, and β-Lactamase Inhibitors Against MRSA

We determined that the observed synergy is not limited to theantibiotics assayed, but can be generalized to their respective β-lactamclasses, by testing MRSA N315 and representative clinical MRSA isolatesagainst other carbapenem/penicillin/β-lactamase inhibitor combinations.We found that treatment of MRSA N315 withimipenem/piperacillin/clavulanate (IM/PI/CV) shows equal or greatersynergism to ME/PI/TZ. Meropenem/amoxicillin/tazobactam (ME/AX/TZ)maintains high synergy in MRSA N315 only (FICI=0.04), with clinical MRSAisolate showing less synergy (FICI=0.55) (Table 2B). MICs for componentsof these substituted triples are all below the mean peak human plasmaconcentrations of these in vivo^(46,47). Similar to ME/PI/TZ, IM/PI/CVshows less-than-additive activity against MSSA ATCC 29213 (FICI=1.14)(Table 2B-C). These results further support the necessity of thepresence of the mecA gene product PBP2a with its attendant allosterismfor synergy, due to lack of synergy of carbapenem/penicillin/β-lactamaseinhibitor combinations in methicillin-susceptible S. aureus.

We also tested the effect of replacing the carbapenem component of thecombination with either a monobactam or a cephalosporin, two otherlater-generation β-lactam derivatives. In contrast to ME/PI/TZ, thetriple combinations aztreonam/piperacillin/tazobactam (AZ/PI/TZ) andcefepime/piperacillin/tazobactam (CP/PI/TZ) (FICI for both=0.33) havelower levels of synergy than PI/TZ alone (FICI=0.22) (Table 2B),possibly because aztreonam (a monobactam) has Gram-negative PBPactivity⁴⁸, while cefepime (a cephalosporin) preferentially targets PBP2over PBP1⁸.

We confirmed the targets of the constituents of ME/PI/TZ by reducing theexpression of PBP1, PBP2, PBP2a or PBP3 using a xylose-inducibleantisense-RNA strategy in the MRSA COL strain background⁴⁹. Whenexpression levels of PBP2a were attenuated, the strain behaved as amethicillin-susceptible S. aureus and was sensitized to all testedβ-lactams (FIG. 6A-C). When meropenem, piperacillin, and tazobactam weretested against the pbpA antisense strain, only meropenem showed largerzones of inhibition under xylose induction, confirming PBP1 as a targetof meropenem (FIG. 6D-E). For the pbp2 antisense strain both meropenemand piperacillin showed increased effectiveness under xylose induction,demonstrating that they each have some activity against PBP2 (FIG.6F-G). We did not observe any effect with the pbp3 antisense strain,consistent with our hypothesis that ME/PI/TZ activity is focused ondisrupting PBP1, PBP2, and PBP2a (FIG. 6H-I). The antisense strains inall cases but that of pbp3 showed sensitization to the triplecombination, underscoring the observed synergy.

Example 3. Lack of Adaptation to Meropenem/Piperacillin/TazobactamOver >10 Days for MRSA N315

Development and spread of resistance can dramatically dampen theeffectiveness and longevity of an antimicrobial therapy. We demonstratedthat ME/PI/TZ suppresses the evolution of resistance in MRSA usingserial passaging in sub-inhibitory antibiotic concentrations of thetriple combination and each of its constituents. To more accuratelymodel a clinical treatment in vitro and in vivo, we applied these drugsat fixed dosages over extended periods as occurs in clinical treatment,not at increasing doses over time. During the 11-day experiment, weobserved no evolution of resistance in MRSA N315 to ME/PI/TZ. Incontrast, we observed resistance evolution against all doublecombinations and single constituents within 1-8 days, consistent withprior work^(23,50) (FIG. 2). Viable cells were observed in allconditions above the initially determined MIC for the doubles andsingles, but not for those conditions at or above the initial MIC forME/PI/TZ. Increases in growth rates over time were noted in all doublesand singles, while the growth rate of N315 in sub-MIC ME/PI/TZ over timewas unchanged throughout the experiment, equivalent to the no-drugcontrol (FIG. 2)²³. Also, N315 exposed to the double combination ME/PIshowed a threefold increase in MIC after day one, indicating that viablecells were present after day one, but did not grow until further passageand adaptation. Determination of the minimal-bactericidal concentration(MBC) confirmed that the triple combination ME/PI/TZ is bactericidalagainst MRSA N315 (Table 4B). Together, these results demonstrate thesuppression of emergence of new resistance against ME/PI/TZ in MRSAN315.

Example 4. Reciprocal Collateral Sensitivities of Components of theseCombinations Underlie Suppression of Adaptation

To determine whether collateral sensitivity was a factor in thesuppression of adaptation of ME/PI/TZ, we analyzed the effects of priorexposure of MRSA N315 to a range of β-lactams on susceptibility to theother components (FIG. 3 and FIG. 7). We observed that there was strongreciprocal collateral sensitivity between meropenem and piperacillin,and between piperacillin and ME/TZ, while PI/TZ sensitized MRSA N315 tomeropenem, but not reciprocally. Collateral sensitivity to piperacillinwas also conferred by prior exposure to tazobactam, but not vice-versa.Interestingly, no collateral sensitivity was found to tazobactam afterexposure to any other single or double compounds. Collateral sensitivityand resistance profiles of amoxicillin and piperacillin are nearlyidentical, with adaptation to meropenem also sensitizing MRSA N315 toamoxicillin (FIG. 3 and FIG. 7). Piperacillin also showed collateralsensitization to imipenem, an even more potent carbapenem against MRSAN315. However, none of the cephalosporins tested for collateralsensitivity by the carbapenem/penicillin/β-lactamase inhibitorcombinations or constituents resulted in sensitivity, but ratherincreased resistance or indifference was noted. These results confirmthat the observed suppression of resistance by collateral sensitivity isspecific to the constituent drug classes of ME/PI/TZ.

Example 5. Adapted MRSA N315 Undergoes Large-Scale Genomic Alterations

We used whole-genome sequencing to investigate the genomic basis of thesensitivity and resistance phenotypes of wild-type and adapted MRSA N315strains. We found no mutations in PBP or β-lactamase genes within any ofthe adapted MRSA N315 isolates. However, absence of read coverageidentified that the penicillinase plasmid pN315 was lost in isolatesadapted to tazobactam-only (100 μg/ml) and ME/TZ (11.1 μg/ml each) (FIG.4A). This plasmid loss occurred much more rapidly than with previouslyreported techniques for curing plasmids from MRSA, such as high heat andSDS treatment⁵¹. In PI/TZ adapted isolates, we observed thatapproximately 400 kb of the MRSA N315 chromosome (GenBank ID:BA000018.3) was duplicated after analysis of read coverage depth, fromapproximate genomic positions 2,100,000 to 2,550,000 bp. Interestingly,this interval contains several putative and confirmed genes involved incell-wall synthesis, including ddlA D-Ala-D-Ala ligase (FIG. 8).

The loss of pN315 in MRSA N315 correlates with increased sensitivity topiperacillin and amoxicillin, both penicillins that should be sensitiveto the blaZ (PC1) class A β-lactamase encoded on the plasmid. However,the loss of pN315 also results in increased resistance totazobactam-only and ME/PI/TZ (FIG. 3, FIG. 7, Table 5A). One possiblelink between the presence of pN315 and ME/PI/TZ activity is the knownregulatory crosstalk between Mecl and Blal repressors and their sharedmec operon target^(52-54.) To test the effect of the loss of pN315 onexpression of genes known to be important for ME/PI/TZ activity, weperformed qRT-PCR analysis of the adapted and wild-type MRSA N315strains (FIG. 4B). We determined that expression of the blaZ β-lactamasein the pN315 plasmid within wild-type MRSA N315 is constitutive, but inclones adapted to tazobactam we saw no expression of blaZ, consistentwith loss of pN315 in these clones. We also found that expression ofmecA is constitutive in the blaZ-null MRSA N315 isolate that was adaptedto tazobactam at 100 μg/ml, consistent with disregulation of the mecoperon via loss of pN315 and the bla operon. Finally, we foundtazobactam to be a strong inducer of mecA in wild-type MRSA N315, atlevels similar to the constitutive expression of mecA seen in theblaZ-null condition.

Example 6. Synergy of ME/PI/TZ when MRSA N315 has Evolved Resistance toConstituents

We then examined the role that resistance to components of ME/PI/TZ hason its effectiveness against MRSA (Table 5A). Previous exposure of MRSAN315 to piperacillin at either 33.3 or 100 μg/ml showed subsequentsensitization of the strain to ME/PI/TZ, from 3.7 to 1.2 μg/ml for eachcomponent. However, prior exposure of MRSA N315 to ME/TZ (11.1 μg/mleach) or meropenem-only (33.3 μg/ml) showed a nine-fold increase inlevels of resistance to ME/PI/TZ (increasing from 3.7 to 33.3 μg/ml foreach component). Exposure to tazobactam-only gave intermediate gains inresistance to ME/PI/TZ up to day 7 (11.1 μg/ml each), and higherresistance at day 11 (33.3 μg/ml each). Prior exposure to ME/PI or PI/TZgenerated only a threefold increase in MIC (from 3.7 to 11.1 μg/ml) overthe 11 days.

Despite the elevated MICs to ME/PI/TZ in the isolates adapted to thecomponent drugs, the triple-drug combination still maintained synergy inall adapted isolates (Table 5B). This is consistent with synergisticdrug activity within the range of ME/PI/TZ MICs observed for the 72clinical MRSA isolates (Table 4), relative to their single-drug MICs.These results show that even when genomic changes enabling sub-componentresistance can be selected, the overall synergistic activity of thetriple-drug combination is maintained. In contrast to recent work with anon-pathogenic E. coli strain³⁶, we observed no change in the overalldrug interaction profile of ME/PI/TZ regarding synergy with increasedresistance to any component drug.

Example 7. ME/PIT/TZ is as Effective as Linezolid Against MRSA In Vivo

Next we tested if ME/PI/TZ or its constituents can be effective intreating MRSA infections in vivo using a neutropenic mouse model ofperitonitis. Blood taken at 11 h post-infection from mice that weretreated with either ME/PI/TZ, ME/PI (67 mg/kg each) or linezolid (30mg/kg) yielded zero plated colonies and no growth in liquid cultures,indicating clearance of infection (FIG. 5, Table 7). All mice(n=6/group) from each of these treatments survived for six dayspost-infection (total duration of the mouse study). The efficacy ofME/PI/TZ and ME/PI was similar to linezolid monotherapy based onclearance of MRSA infection and survival of all treated mice compared tovehicle (p=0.02, Fisher's exact test).

In contrast to the complete rescue of the infected mice by ME/PI/TZ,ME/PI, or linezolid, several mice treated with ME/TZ, PI/TZ, ormeropenem-alone, and all mice treated singly with piperacillin ortazobactam succumbed to the infection, most within 48 h (FIG. 5, Table7). Treatment with these other drug regimens was not significantlydifferent than treatment with vehicle (p>0.05, Fisher's exact test)(Table 6A), where all mice also succumbed to the infection within 48 h.

We tested MRSA N315 cultures from blood drawn from mice treated withmeropenem, piperacillin, or vehicle for their in vitro MICs againstME/PI/TZ and its constituent single drugs to determine whetheradaptation occurred during passage in vivo. All four tested isolates ofMRSA N315 had identical MICs for the triple ME/PI/TZ and all constituentdrugs, and thus identical synergy (Table 6B). These data suggest noadaptation occurred within these strains to overcome the triple ME/PI/TZtested within the 11-h passage in vivo.

Discussion for the Examples.

We have shown that triple antibacterial combinations containingcarbapenems, penicillins, and β-lactamase inhibitors target multiplenodes in the same cellular system (cell-wall synthesis) and are highlysynergistic and bactericidal against diverse MRSA strains in vitro, atclinically achievable concentrations. This contrasts with recent workshowing collateral sensitivity and synergy to arise from combinations ofdrug classes working against orthogonal cellular targets innon-pathogenic lab strains only^(25,36). Because carbapenems and otherdrugs at high concentration could have toxic effects, reduced per-drugdosages via synergy mitigate potential toxicities⁵⁵. Our 3-Dcheckerboard testing confirmed the optimal input concentrations forME/PI/TZ to be given in a 1:1:1 ratio (2 μg/ml each) against MRSA N315,which is below the susceptibility breakpoints for these compoundsagainst methicillin-susceptible S. aureus and is an 8-to-64-foldreduction in input concentrations for these formerly inactive drugsagainst this highly resistant MRSA strain. Our mechanistic analysessupport our hypothesis that targeting of PBP1 by meropenem, targeting ofPBP2 by piperacillin, protection of piperacillin by tazobactam fromβ-lactamase cleavage, and allosteric opening of the active site of PBP2aby meropenem for inhibition by another molecule of antibiotic in thecombination, result in synergy by simultaneously perturbing multiplecomponents of the MRSA cell-wall synthesis system (FIG. 9).

We have also preliminarily shown that this combination has activity in ahighly lethal neutropenic MRSA in vivo model, demonstrating that thistriple combination of clinically approved β-lactams can clear infectionsimilar to substantially more expensive monotherapies like linezolid.The plasma levels of meropenem observed in mice correlate well withplasma drug levels in healthy humans⁵⁶, and meropenem would attain theKd at these clinically achievable concentrations to trigger allosteryfor opening of the active site of PBP2a, providing accessibility forinhibition by meropenem and other β-lactams in the combination^(9,10).

Notably, the double combination ME/PI cleared the MRSA N315 infection invivo similarly to ME/PI/TZ and linezolid within 11 h. In vitro weobserved high synergy scores and reciprocal collateral sensitivity forthis combination, similar to what was seen for ME/PI/TZ, but ME/PI didnot suppress evolution of resistance to the same extent that ME/PI/TZdid. This property may not have been relevant to this aggressiveinfection model, but may be important for longer treatment times seen inhuman infections with MRSA. ME/PI/TZ is also likely to be effective atlower total concentrations than ME/PI because of its higher synergy.Longer exposure of the N315 strain to the tazobactam component ofME/PI/TZ in vivo may also promote ejection of pN315 plasmid withconcomitant sensitization to the penicillin component, in line with thein vitro results for collateral sensitivity and suppression ofadaptation. Indeed, to more adequately address this question, potentiallonger-term in vivo resistance evolution would need to be tested undersub-lethal concentrations of the drugs in important follow-up mouseexperiments.

Our robust mechanistic in vitro results and preliminary in vivo resultsfor ME/PI/TZ activity suggest this combination may be made immediatelyavailable for use in the clinic, since it includes currentlyFDA-approved drugs, which had met their obsolescence as monotherapiesagainst MRSA decades ago. However, further mechanistic features of thecombination that were shown in vitro (synergy, resistance suppressionover longer periods of dosing, collateral sensitivity, etc.) willrequire substantially more in vivo testing to support the promising butpreliminary activity observed in our highly aggressive neutropenic mousemodel.

We note that high resistance to meropenem or tazobactam slightly reducesthe effectiveness of ME/PI/TZ, while maintaining its synergy, and ourresistance evolution analysis cannot account for resistance genesacquired horizontally that could break the relationship betweenmeropenem, piperacillin, and tazobactam. Despite these caveats, webelieve the ME/PI/TZ combination is an immediately viable anti-MRSAtherapeutic, and endorse further mechanistic exploration into theputative superior efficacy of high-order antibiotic combinations thatare both synergistic and encoded by collaterally sensitive constituents.Having similar activity to linezolid against MRSA in vivo, the potentialefficacy of ME/PI/TZ reopens broad prospects for the clinical use ofβ-lactams against the staphylococci. It also suggests that this line ofresearch into repurposing existing antibiotics in carefully designedsynergistic combinations would address immediate clinical needs, asthese agents are already approved for human use. Emergence of resistanceto any antibiotic or any antibiotic combination is inevitable. Yet, asevidenced in our study, combinations composed of key drug-druginteraction features may be a tool in mitigating the emergence ofantibiotic resistance by preserving the usefulness of existing agentsavailable to us in our pharmacological armamentarium.

Methods for the Examples

Microbiological Studies:

MRSA N315 was a gift from Dr. Steven Gill, University of Rochester,Rochester, N.Y., USA. S. aureus ATCC 29213 was acquired from theAmerican Type Culture Collection. De-identified clinical MRSA isolateswere selected at random from the clinical isolate strain bank atBarnes-Jewish Hospital, St. Louis, Mo., USA. Minimal inhibitoryconcentration (MIC) assays for inhibition of growth were performedfollowing the recommendations of the Clinical and Laboratory StandardsInstitute (CLSI)⁴³. Briefly, 23 antibacterial compounds (SupplementalTable 1) were selected based on coverage of all major drug classes,including three compounds not classified as antibiotics for human use,but with known antibacterial properties. Compounds were dissolved indimethyl sulfoxide (DMSO) to a stock concentration of 50 mg/ml.Exceptions: Sulfometuron at 20 mg/ml in DMSO; Tobramycin, D-cycloserine,and colistin at 50 mg/ml in H2O and filtered at 2 μm. The 23 compoundswere formulated into all 253 possible unique pairwise combinations atfixed ratios and at 100× concentrations in solvent. To increase therange of concentrations assayed for possible synergistic or antagonisticdrug interactions (>2,000-fold), the drug stocks were arrayed intothreefold dilution series down eight rows in 96-well Costar master drugplates, using a BioMek FX robotic liquid handler (Beckman Coulter,Inc.). Drugs were then mixed 1:100 into 96-well plates containing 200μl/well of cation-adjusted Mueller-Hinton broth (CAMHB). All drugsusceptibility assay wells were inoculated with ˜1 μl of mid-log phasebacterial culture at 0.5 McFarland standard (˜2×10⁸ CFU/ml) and grown at37° C. for 24 h. Endpoint growth at 37° C. after 24 h was determined byoptical density at 600 nm using a Synergy H1 reader (BioTek, Inc.).

Synergy of antibiotic combinations was determined using the fractionalinhibitory concentration index (FICI) method 57,58. By this method, theMIC of the antibiotic compound in combination is divided by the MIC ofthe compound alone, yielding the fractional contribution of each drugcomponent in the combination. Quotients for all in a combination aresummed and drug interactions scored using the formula: FICI=(MICA_(comb A B C))/MIC_(agent A+)(MIC B_(comb A B C))/MIC_(agent B+)(MICC_(comb A B C))/MIC_(agentC) Select pairwise combinations against MRSAwere then combined with each of the 21 remaining single drugs to maketriple combinations, formulated and tested in identical fashion to thedouble combinations. Synergy of combinations was confirmed viatriplicate measurements of drug conditions at the MIC. Based on its highsynergy against MRSA N315 in the sparse screening, ME/PI/TZ and itsconstituents were selected for further characterization. Finalsusceptibility testing of ME/PI/TZ and its components was performedusing twofold dilution from 128 to 2 μg/ml for each component

Minimal bactericidal concentration (MBC) for ME/PI/TZ in MRSA N315 wasdetermined via duplicate wells of ME/PI/TZ at indicated concentrationsin CAMHB media, inoculated with ˜5×10⁵ CFU/ml of MRSA N315 in mid-logphase and incubated at 37° C. for 24 h. 100 μl of a 1:100 dilution of 50μl drawn from duplicate ME/PI/TZ wells was plated on Mueller-Hinton agar(MHA) plates and incubated overnight for 24 h. No colony growth at ortwo dilutions above the MIC confirmed bactericidal activity, as definedby CLSI⁵⁹. Meropenem (CAS 96036-03-2) and clavulanate (CAS 61177-45-5)were obtained from AK Scientific, Inc. (Union City, Calif., USA).Piperacillin (CAS 59703-84-3), tazobactam (CAS 89786-04-9), imipenem(CAS 74431-23-5), and amoxicillin (CAS 26787-78-0) were obtained fromSigma-Aldrich Co. (St. Louis, Mo., USA).

Adaptation and Cross-resistance Assays:

414 MRSA N315 was grown in 150 μl/well of CAMHB with constant shaking at37° C. and passaged over 11 days in identical 96-well plates containingreplicate threefold dilutions of ME/PI/TZ, ME/PI, ME/TZ, PI/TZ, ME, PI,and TZ. Top concentrations of drug combinations were 33.3 μg/ml for eachcomponent, while top concentrations for single drugs was 100 μg/ml. Totest for cell viability, at the end of the assay on day 11, all wellsfrom the plate were pinned with a sterile 96-pin replicator andtransferred to CAMHB only. After passage, plates were filled 1:1 with30% CAMHB/glycerol and frozen at −80° C. for later analysis.

Growth rate of isolates over passages in each condition was determinedby linear best-fit of logarithm-converted exponential growth phase.Following Hegreness et al^(23,) those wells containing cells in drugconditions whose growth rates were >0.2 h⁻¹ between day one and theaverage of the last six days of growth were considered significantlyadapting to conditions and an adaptation rate α was generated. Adaptedisolates were retrospectively chosen from each combination or singlecompound in wells showing an increase in MIC or growth rate, frozenisolates were streaked out on agar plates to obtain single colonies,re-grown in broth conditions identical to those in which they greworiginally, and then re-inoculated in sterile 96-well plates identicalto the original 11-day 431 plates.

Expression Profiling with qRT-PCR:

Wild-type and adapted MRSA N315 isolates were grown in triplicate in 100ml flasks to mid-log phase in CAMHB+/− piperacillin at 11.1 μg/ml ortazobactam at 33.3 μg/ml. To harvest cells at mid-log phase, eachculture flask was split into 2×50 ml screw-cap tubes, spun down at 4° C.for 10 min at 3500 rpm, supernatant removed, and pellets combinedcarefully with a 2 ml serological pipette. 1 ml RNAprotect BacteriaReagent (Qiagen, Valencia, Calif., USA) was added to pellets tostabilize the RNA, vortexed briefly, and incubated for 5 min at RT.After incubation, tubes were spun again at 4° C. for 10 min at 3500 rpm,supernatant removed, and the pellets were stored at −80° C. Total RNAwas extracted by the following protocol:

-   -   (1) Resuspend cell pellets in 500 μl Buffer B (200 mM NaCl, 20        mM EDTA).    -   (2) Add 210 μl 20% SDS.    -   (3) Add ˜250 μl volume of acid-washed sterile glass beads        (Sigma, Inc.).    -   (4) Add 500 μl Phenol:Chloroform:IAA.    -   (5) Bead beat on ‘high’ for 5 min.    -   (6) Spin at 8000 rpm at 4° C. for 3 min (to separate the        phases).    -   (7) Remove top aqueous phase and transfer into a new tube.    -   (8) Add 700 μl isopropanol.    -   (9) Add 70 μl 3M NaOAc, mix thoroughly by inversion.    -   (10) Spin at 4° C., max rpm, for 10 min.    -   (11) Aspirate supernatant.    -   (12) Add 750 μl ice cold 70% EtOH, spin at max rpm at 4° C. for        5 min.    -   (13) Aspirate supernatant, to let the EtOH dry, leave tubes open        in RNase free area.    -   (14) Add 100 μl nuclease free water to each tube and resuspend        (put tubes in 50° C. heat block, vortexing periodically).    -   (15) Add 12 μl TURBO-DNase buffer (Ambion, Inc.) and 10 μl        RNase-free TURBO-DNase to each sample, and incubate at 37° C.        for 30 min.    -   (16) Purify samples using MEGAClear columns and kit per        manufacturer protocol.    -   (17) Re-purify samples using Baseline-ZERO DNase buffer        (Epicentre, Inc.) and 10 μl Baseline-ZERO DNase, following        manufacturer protocol.    -   (18) Elute final RNA samples with 30 μl TE buffer, pH 7.0.

First-strand cDNA was synthesized from total RNA with SuperScriptFirst-Strand Synthesis System for RT-PCR (Life Technologies, Carlsbad,Calif., USA). qRT-PCR of pbp2, mecA and blaZ in MRSA N315 was performedagainst gyrB using SYBR Select Master Mix for CFX (Life Technologies,Carlsbad, Calif., USA) on a CFX96 Real-Time PCR Detection System(Bio-Rad Laboratories, Inc, Hercules, Calif., USA). Primer sequencesused (0.3 μM each):

pbp2_F: SEQ ID NO: 1 CGTGCCGAAATCAATGAAAGACGC, pbp2_R: SEQ ID NO: 2GGCACCTTCAGAACCAAATCCACC; mecA_F: SEQ ID NO: 3 TGGAACGATGCCTATCTCATATGC,mecA_R: SEQ ID NO: 4 CAGGAATGCAGAAAGACCAAAGC; blaZ_F: SEQ ID NO: 5TTTATCAGCAACCTTATAGTCTTTTGGAAC, blaZ_R: SEQ ID NO: 6CCTGCTGCTTTCGGCAAGAC, gyrB_F: SEQ ID NO: 7 CGATGTGGATGGAGCGCATATTAG,gyrB_R: SEQ ID NO: 8 ACAACGGTGGCTGTGCAATATAC.

CFX protocol: 2 min @ 50° C., 2 min @ 95° C., (15 s @ 95° C., 1 min @60° C.)×40 cycles. Gene expression was determined using the ΔΔCt methodof normalized quantitation 60, where Ct indicates the cycle number atwhich exponential growth phase increases above threshold fluorescencesignal.

Sequencing Library Preparation:

Genomic DNA (gDNA) was extracted from wild-type and adapted MRSA N315using lysostaphin digestion and phenol:chloroform:IAA extraction asfollows:

-   -   (1) Draw 1 ml aliquots from overnight 5 ml shaking cultures        of S. aureus strains, spin down at 13,000 rpm for 3 min, pour        off media, add additional 1 ml of culture and repeat.    -   (2) Add 500 μl of 2× Buffer A (NaCl 200 mM, Tris 200 mM, EDTA 20        mM) at 4° C. to pelleted cells and vortex briefly to resuspend        cells.    -   (3) Add 2.5 μl of 10 mg/ml (200×) lysostaphin (Sigma-Aldrich,        Inc.) to tubes.    -   (4) Flick mix and spin down tubes, place in 37° C. dry bath for        1 h.    -   (5) Fast cool micro-centrifuge to 4° C.    -   (6) Add ˜250 μl of 0.1 mm zirconium beads (BioSpec Products, cat        #1107910).    -   (7) Add 210 μl of 20% SDS.    -   (8) Add 500 μl phenol:chloroform:IAA (25:24:1, pH 7.9), chill        samples on ice.    -   (9) Bead beat on the “homogenize” setting for 4 min (beat 2 min,        ice 2 min, beat 2 min).    -   (10) Spin at 6800 rcf (4° C.) for 3 min.    -   (11) Spin down PLG columns (5Prime, cat #2302820) at max speed        (20,800 rcf) for 30 s at RT while waiting.    -   (12) Transfer aqueous phase (˜500 μl) to pre-spun phase-lock gel        tube.    -   (13) Add equal amount (500 μl) of phenol:chloroform:IAA        (25:24:1, pH 7.9) to tube and mix by inversion (DO NOT VORTEX).    -   (14) Spin tubes at max speed (20,800 rcf) (RT) for 5 min.    -   (15) Transfer aqueous phase (˜500 μl) to a new Eppendorf tube.    -   (16) Add 500 μl of −20° C. isopropanol.    -   (17) Add 50 μl (1/10 vol.) of 3M NaOAc at pH 5.5 (Ambion,        AM9740), and mix thoroughly by inversion.    -   (18) Store at −20° C. for at least 1 h (overnight is preferable        but not necessary).    -   (19) Spin at max speed at 4° C. for 20 min.    -   (20) Wash pellet with 500 μl of 100% EtOH (RT) and spin down at        4° C. for 3 min.    -   (21) Carefully pipet off EtOH, air-dry>15 min.    -   (22) Add 30 μl of TE (Ambion, AM 9861), incubate at 50° C. for 5        min.    -   (23) Run DNA through QIAGEN QIAQuick PCR purification column        with the following modifications: RNase A treatment at beginning        of column clean-up. Combine 4 μl Qiagen RNase (100 mg/ml) with        every 300 μl buffer PB used, incubate in buffer PB/RNase for 15        min at RT.    -   (24) Let PE wash buffer sit in column at RT for 2 min, elute        gDNA with 35 μl of EB buffer pre-heated to 55° C., letting sit        for 1 min before final spin.

We sheared 500 ng of total DNA from each genome to ˜300 bp fragments innine rounds of shearing of ten min each on the BioRuptor XL. In eachround the power setting was ‘H’ and samples were treated for 30 s andallowed to rest for 30 s. Each sample was concentrated using the QiagenMinElute PCR purification kit per the manufacturer's protocol. EndRepair of the sheared DNA fragments was initiated with the addition of2.5 μl of T4 DNA ligase buffer with 10 mM ATP (NEB, B0202S), 1 μl of 1mM dNTPs (NEB), 0.5 μl T4 Polymerase (NEB, M0203S), 0.5 μl T4 PNK (NEBM0201S), and 0.5 μl Taq Polymerase (NEB, M0267S). This mixture wasincubated at 25° C. for 30 min, then at 75° C. for 20 min. Barcodedadapters were then added to the solution along with 0.8 μl of T4 DNAligase (NEB, M0202M), for the purpose of ligating the adapters to theDNA fragments. This solution was then incubated at 16° C. for 40 min,then 65° C. for 10 min. The adapter-ligated DNA was then purified usingthe Qiagen MinElute PCR purification kit per the manufacturer'sprotocol.

The DNA fragments were then size selected on a 2% agarose gel in 1×TBEbuffer stained with Biotium GelGreen dye (Biotium). DNA fragments werecombined with 2.5 μl 6× Orange loading dye before loading on to the gel.Adapter-ligated DNA was extracted from gel slices corresponding to DNAof 250-300 bp using a QIAGEN MinElute Gel Extraction kit per themanufacturer's protocol. The purified DNA was enriched by PCR using 12.5μl 2× Phusion HF Master Mix and 1 μl of 10 μM Illumina PCR Primer Mix ina 25 μl reaction using 1 μl of purified DNA as template. DNA wasamplified at 98° C. for 30 s followed by 18 cycles of 98° C. for 10 s,65° C. for 30 s, 72° C. for 30 s with a final extension of 5 min at 72°C. The DNA concentration was then measured using the Qubit fluorometerand 10 nmol of each sample (up to 106 samples per lane of sequencing)were pooled. Subsequently, samples were submitted for IlluminaHiSeq-2500 Paired-End (PE) 101 bp sequencing at GTAC (Genome TechnologyAccess Center, Washington University in St. Louis) at 9 pmol per lane.

DNA Sequence Analysis: Alignment and Variant Calling.

For the wild-type and adapted MRSA N315, all sequencing reads for eachgenome were de-multiplexed by barcode into separate genome bins. Readswere quality trimmed to remove adapter sequence and bases on either endwith a quality score below 19. Any reads shorter than 31 bp afterquality trimming were not used in further analysis. All reads weremapped to the Staphylococcus aureus subsp. aureus N315 chromosome(GenBank ID: BA000018.3) and pN315 plasmid (GenBank ID: AP003139)(command:bowtie2-x<reference_genome_index_name>-1<forward_read_file>-2<reverse_read_file>-q--phred33--very-sensitive-local-I200-X 1000-S<sam_output>). Variants from the reference were called usingsamtools⁶¹ commands: samtools view-buS<sam_file>|samtools sor-m4000000000-<sample_prefix>### samtools index<bam_file>### samtoolsmpileup-uD-f reference_genome> <bam_file>|bcftools view-bcv-><bcf_file>### bcftools view<bcf_file>). The variant call format (VCF)file was then filtered to remove SNPs with a quality score lower than 70or coverage greater than twice the average coverage expected per base.Absence of read coverage or overabundant read coverage indicated plasmidloss or large duplication respectively. Any variant position found fromthe wild-type alignment was determined to be a result of alignment erroror to be derived from lab specific drift in N315 and was removed fromall other VCF files. Each variant position was then compared to knownORF locations in N315 to search for causal variants.

In Vivo Mouse Model of MRSA Infection: Animals.

Outbred ICR female mice (6-8 weeks old, 17-25 g body weight; HarlanLaboratories, Inc., Indianapolis, Ind., USA) were used. Mice were givenTeklad 2019 Extruded Rodent Diet (Harlan Laboratories, Inc.,Indianapolis, Ind., USA) and water ad libitum. Mice were maintained inpolycarbonate shoebox cages containing corncob (The Andersons, Inc.,Maumee, Ohio, USA) and Alpha-dri (Shepherd Specialty Papers, Inc.,Richland, Mich., USA) bedding under 12-h light/12-h dark cycle at 22±1°C. All procedures involving animals were approved by the University ofNotre Dame Institutional Animal Care and Use Committee.

Neutropenic Mouse Peritonitis Model of MRSA Infection.

Doses of cyclophosphamide (100 μl of 50 mg/ml in 0.9% salinecorresponding to 200 mg/kg; Alfa Aesar, Ward Hill, Mass., USA) weregiven intraperitoneally (IP) at 4 days and 1 day prior to infection. TheS. strain N315 was streaked onto Brain-Heart Infusion (BHI; BectonDickson and Company, Sparks, Md., USA) agar and grown overnight at 36°C. The MRSA N315 bacterial inoculum was adjusted to approximately 1×10⁸CFU/ml (corresponding to OD₅₄₀=0.5), then diluted to give 2×10⁷ CFU/ml.A 10% porcine mucin (Sigma-Aldrich, St. Louis, Mo., USA) suspension wasprepared and adjusted to pH 7. Immediately prior to infection, thebacterial inocula were diluted 1:1 with 10% mucin to a finalconcentration of 1×10⁷ CFU/ml in 5% mucin. The mice were then infectedIP with 0.5 ml of this inoculum. In vivo dosing of compounds in mice wascompared with mean or range peak human plasma concentrations of studiedβ-lactams^(44,46,47,62,63).

Antibiotic Preparation.

Meropenem was obtained from AK Scientific, Inc. (Union City, Calif.,USA), piperacillin and tazobactam were obtained from Sigma-Aldrich Co.(St. Louis, Mo., USA). Linezolid (CAS 165800-03-3) was obtained fromAmplaChem (Carmel, Ind., USA). Antibiotics were dissolved at aconcentration of 16.67 mg/ml in 30% DMSO/30% propylene glycol/40% water.Linezolid was used as positive control and was prepared at 7.5 mg/ml.Vehicle (30% DMSO/30% propylene glycol/40% water) was included asnegative control. The dosing formulations were sterilized by passingthrough 0.2 μm filter prior to injection.

Bacterial Isolation from Blood.

Blood samples were checked for bacterial growth by plating and liquidculture. Whole blood (100 μl, three samples per group) was spread ontoBrain-Heart Infusion (BHI) agar plates and incubated at 36° C.overnight. Colonies were counted and three colonies were selected, grownovernight in liquid BHI culture at 36° C., then mixed 1:1 with 30%LB-glycerol and stored at −80° C. The remaining three blood samples ofeach group (50 μl) was added to 5 ml BHI broth and incubated overnightat 36° C. When growth was noted, cultures were mixed 1:1 with 30%LB-glycerol and stored at −80° C.

Statistical Analyses:

Data for minimal inhibitory concentrations (MICs) are derived fromtriplicate measurements. Adaptation data are taken from two replicateexperiments for each drug combination condition. Data for qRT-PCRexpression profiling are derived from three replicate experiments takenfrom three biological replicates each, with standard error ofmeasurement calculated. Mice were treated in groups of six, and growthdetermination of bacteria determined via plate and broth culture intriplicate. Fisher's Exact test with Bonferroni correction was used for8 independent tests (comparing each treatment to 610 vehicle).

TABLE 1 23 antibacterial compounds used to formulate combinations inthis study. MIC in MRSA Target mechanism N315 Compound in bacteriaAntibiotic Class (μg/ml) Sulfamethoxazole Folic acid pathway Sulfonamide100 Trimethoprim Folic acid pathway Pyrimidine derivative 6.2Levofloxacin DNA synthesis Fluoroquinolone 0.4 Bleomycin DNA synthesisGlycopeptide >500 Gemfibrozil Lipid synthesis *Fibrate >200(hyperlipidemia agent) Sulfometuron Amino acid *Broad-spectrum urea >200biosynthesis herbicide Disulfiram Osmotic stress *Thiuram disulfide 11.1response (anti-alcohol therapeutic) Tigecycline Protein synthesisTetracycline 0.4 Mupirocin Protein synthesis Pseudomonic acid 0.4Linezolid Protein synthesis Oxazolidinone 3.7 Azithromycin Proteinsynthesis Macrolide >200 Clindamycin Protein synthesis Lincosamide >500Chloramphenicol Protein synthesis Amphenicol 11.1 Tobramycin Proteinsynthesis Aminoglycoside >500 Rifampin Transcription Rifamycin 0.4Vancomycin Cell wall synthesis Glycopeptide 0.4 Piperacillin Cell wallsynthesis β-lactam/Penicillin 64 (Penam)/Broad- spectrum Aztreonam Cellwall synthesis β-lactam/ >500 Monobactam/Gram- negative specificCefepime Cell wall synthesis β-lactam/ 100 Cephalosporin 4^(th)generation (Cephem)/Broad- spectrum Meropenem Cell wall synthesisβ-lactam/ 16 Carbapenem/Ultra- broad-spectrum Tazobactam Cell wallsynthesis β-lactamase inhibitor 128 (Penam) D-Cycloserine Cell wallsynthesis Analogue of the 56 amino acid D-alanine Colistin Cell membranelysis Polymyxin 500 Compounds are grouped by target mechanism of action.*Compound not formally classified as an antibiotic drug, but has knownantibacterial properties.

TABLE 2 Fractional Inhibitory Concentration Index (FICI) profiling ofcombinations. A. Interpretive criteria for FICI scoring. FICIInterpretation ≤0.5 Synergy >0.5 to <1.0 Partial Synergy   1.0Additivity >1.0 to <4.0 Indifference ≥4.0 Antagonism Combination ME/ CP/AZ/ ME/ ME/ IM/ in stream PI/TZ PI/TZ PI/TZ AX/TZ AX/CV PI/CV ME/PIME/TZ PI/TZ IM/PI IM/CV PI/CV B. FICI profiles of various triplecombinations of carbapenems/penicillins/β-lactamase inhibitors againstMRSA and MSSA strains. MRSA N315 0.11 0.33 0.33 0.04 0.41 0.06 0.44 0.670.22 0.15 0.67 0.44 SCCmec type II MRSA #181 0.28 ND ND 0.55 ND 0.11 NDND ND ND ND ND SCCmec type II MSSA ATCC 1.12 ND ND ND ND 1.14 2.97 8.610.36 1.11 1.04 0.43 29213 C. MIC profiles of same combinations (μg/ml)Constituent double combinations are shown for comparison. MRSA N315   2each 11.1 each 11.1 each  0.4 each 3.7 each 0.12/1.2/1.2 2/4 8/2 16/20.37/3.7 1.11/1.11 1.11 each SCCmec type II MRSA #181 11.1 each ND ND11.1 each ND 0.37/3.7/3.7 ND ND ND ND ND ND SCCmec type II MSSA ATCC0.27 each ND ND ND ND 0.04/0.4/0.4 0.4 each 1.2 each 1.2 each 0.04/0.40.04/0.4  1.2 each 29213

TABLE 3 Compiled FICI data for ME/PI/TZ against MRSA N315 and 72clinical MRSA isolates. A-B. 72 clinical BRSA isolates (with SCCmectype, if known) and FICI scores for ME/PI/TZ against 72 clinical MRSAisolates. A B Clinical MRSA Clinical MRSA isolate (SCCmec isolate(SCCmec FICI score type) FICI score type) (Continued) (Continued)  40.22 124 0.28  7 0.22 131 (II) 0.37  13 0.15 132 0.22  15 0.5 140 0.22 22 0.22 144 0.15  25 0.22 146 0.22  27 0.67 150 0.67  31 0.15 152 0.22 35 (II) 0.17 155 0.39  37 0.15 161 0.37  39 (II) 0.67 163 0.37  41 (II)0.09 164 0.17  45 0.22 165 0.15  48 0.15 167 0.22  53 0.22 168 0.22  590.07 169 0.17  64 (II) 0.44 171 0.17  66 0.11 172 0.67  70 0.67 175 0.15 72 0.15 177 0.22  73 (IV) 0.34 181 (II) 0.28  74 0.34 182 0.5  75 0.34189 0.15  77 (II) 0.67 190 0.34  85 0.5 193 (II) 0.34  89 0.07 194 0.15 90 0.22 195 0.15  95 0.22 197 0.15  99 0.44 200 0.15 101 0.22 201 0.44103 0.37 204 0.39 104 (II) 0.15 205 0.22 109 0.17 206 0.22 118 0.07 2130.44 121 0.22 217 0.15 122 0.22 219 0.15

TABLE 4 Compiled MIC and MBC data for ME/PI/TZ against MRSA isolates. A.Distribution of MIC resistance profiles of studied MRSA isolates againstME/PI/TZ. MIC of ME/PI/TZ Components (μg/ml) # of MRSA isolates % oftotal 33.3 9 12.3 11.1 27 36.9 3.7 27 36.9 1.2 8 10.9 0.4 2 2.7 Total 73— B. Confirmation of minimum bactericidal concentration (MBC) forME/PI/TZ in MRSA N315. Plate Concentrations Colonies Plate A ColoniesPlate B ME/PI/TZ 2/2/2 Punctate lawn Too many to count ME/PI/TZ 4/4/4*40 0 ME/PI/TZ 8/8/8 2 0 ME/PI/TZ 16/16/16 0 0 ME/PI/TZ 32/32/32 8 0 *MIC= 4/4/4 μg/ml

TABLE 5 Change in ME/PI/TZ resistance phenotype of MRSA N315 over 11days after repeated exposure to constituents of ME/PI/TZ. A. Isolateswere selected on days when an increase in MIC or growth rate was noted.Antibacterial concentrations (listed in μg/ml) show the adaptationconditions for MRSA N315. Post-adaptation MICs to each component ofME/PI/TZ are shown in selected isolates versus passage day. Passage day/ME/PI ME/T7 PI/TZ Adaptation 11.1 μg/ml 11.1 μg/ml 3.7 μg/ml MeropenemPiperacillin Piperacillin Tazobactam conditions each each each 33.3μg/ml 100 μg/ml 33.3 μg/ml 100 μg/ml 1 no change 11.1/11.1/11.11.2/1.2/1.2 11.1/11.1/11.1 no change 1.2/1.2/1.2 11.1/11.1/11.1 2 nochange no change no change no change 3.7/3.7/3.7 3.7/3.7/3.711.1/11.1/11.1 3 no change no change no change no change no change nochange no change 4 no change no change no change no change no change nochange no change 5 no change 33.3/33.3/33.3 no change no change nochange no change no change 6 no change no change 11.1/11.1/11.1 nochange no change no change no change 7 11.1/11.1/11.1 no change nochange 33.3/33.3/33.3 no change no change 11.1/11.1/11.1 8 no change nochange no change no change 1.2/1.2/1.2 1.2/1.2/1.2 no change 9 no changeno change no change no change no change no change no change 10 no changeno change no change no change no change no change no change 1111.1/11.1/11.1 33.3/33.3/33.3 11.1/11.1/11.1 33.3/33.3/33.3 1.2/1.2/1.21.2/1.2/1.2 33.3/33.3/33.3 B. FICI of MRSA N315 against ME/PI/TZ afteradaptation components in vitro. MRSA N315 isolate adapted to: ME/PIME/TZ PI/TZ ME PI TZ FICIME/PI/TZ 0.22 0.83 0.22 0.83 0.05 0.83

TABLE 6 A. Statistics of in vivo treatments with β-lactams Aftermultiple hypothesis Drug condition tested p-value versus vehiclecorrection (Bonferroni) ME 0.6006 4.848 (1) PI 1    8 (1) TZ 1    8 (1)ME/PI 0.0022 0.0176 ME/TZ 0.0152 0.1216 PI/TZ 0.606 4.848 (1) ME/PI/TZ0.0022 0.0176 Linezolid 0.0022 0.0176 B. In vitro MICs and FICI scoresfor MRSA N315 after passage in vivo under indicated drug conditions.Colonies from mice given: Wild-type MIC (μg/ml) for: ME PI Vehicle N315ME/PI/TZ 3.7/3.7/3.7 3.7/3.7/3.7 3.7/3.7/3.7 3.7/3.7/3.7 ME 33.3 33.333.3 33.3 PI 33.3 33.3 33.3 33.3 TZ 100 100 100 100 FICI 0.26 0.26 0.260.26

TABLE 7 Data from Animal Experiments MRSA Bacterial Time of Compounddose every 2nd Cheek 3rd 4th 5th 6th detection ID Inf (Dose level) 8 hrsSC dose SC Bleed dose SC dose SC dose SC dose SC results Result 1 7:30AM meropenem 8:30 AM 4:30 PM 6:31 PM 12:31 AM  8:30 AM 4:30 PM 12:31 AM Plated: Dead 4/6 (67 mg/kg) lawn of bacteria 2 7:31 AM meropenem 8:31 AM4:31 PM 6:32 PM 12:32 AM  8:31 AM 4:31 PM 12:32 AM  Plated: 11 Alive (67mg/kg) colonies 3 7:32 AM meropenem 8:32 AM 4:32 PM 6:33 PM 12:33 AM 8:32 AM 4:32 PM 12:33 AM  Plated: 3 Alive (67 mg/kg) colonies 4 7:33 AMmeropenem 8:33 AM 4:33 PM 6:34 PM 12:34 AM  8:33 AM 4:33 PM 12:34 AM Liquid Alive (67 mg/kg) culture: no growth 5 7:34 AM meropenem 8:34 AM4:34 PM 6:35 PM 12:35 AM  8:34 AM 4:34 PM 12:35 AM  Liquid Dead (67mg/kg) culture: growth 6 7:35 AM meropenem 8:35 AM 4:35 PM 6:36 PM 12:36AM  8:35 AM 4:35 PM 12:36 AM  Liquid Alive (67 mg/kg) culture: no growth7 7:40 AM piperacillin 8:40 AM 4:40 PM 6:41 PM 12:41 AM  8:40 AM 4:40 PM12:41 AM  Plated: Dead 0/6 (67 mg/kg) lawn of bacteria 8 7:41 AMpiperacillin 8:41 AM 4:41 PM 6:42 PM 12:42 AM  8:41 AM 4:41 PM 12:42 AM Liquid Dead (67 mg/kg) culture: growth 9 7:42 AM piperacillin 8:42 AM4:42 PM 6:43 PM 12:43 AM  8:42 AM 4:42 PM 12:43 AM  Plated: 40 Dead (67mg/kg) colonies 10 7:43 AM piperacillin 8:43 AM 4:43 PM 6:44 PM 12:44AM  8:43 AM 4:43 PM 12:44 AM  Liquid Dead (67 mg/kg) culture: growth 117:44 AM piperacillin 8:44 AM 4:44 PM 6:45 PM 12:45 AM  8:44 AM 4:44 PM12:45 AM  Liquid Dead (67 mg/kg) culture: no growth 12 7:45 AMpiperacillin 8:45 AM 4:45 PM 6:46 PM 12:46 AM  8:45 AM 4:45 PM 12:46 AM Plated: Dead (67 mg/kg) lawn of bacteria 13 7:50 AM tazobactam 8:50 AM4:50 PM 6:51 PM 12:51 AM  8:50 AM 4:50 PM 12:51 AM  Plated: Dead 0/6 (67mg/kg) lawn of bacteria 14 7:51 AM tazobactam 8:51 AM 4:51 PM 6:52 PM12:52 AM  8:51 AM 4:51 PM 12:52 AM  Liquid Dead (67 mg/kg) culture:growth 15 7:52 AM tazobactam 8:52 AM 4:52 PM 6:53 PM 12:53 AM  8:52 AM4:52 PM 12:53 AM  Plated: Dead (67 mg/kg) lawn of bacteria 16 7:53 AMtazobactam 8:53 AM 4:53 PM 6:54 PM 12:54 AM  8:53 AM 4:53 PM 12:54 AM Liquid Dead (67 mg/kg) culture: growth 17 7:54 AM tazobactam 8:54 AM4:54 PM 6:55 PM 12:55 AM  8:54 AM 4:54 PM 12:55 AM  Plated: Dead (67mg/kg) lawn of bacteria 18 7:55 AM tazobactam 8:55 AM 4:55 PM 6:56 PM12:56 AM  8:55 AM 4:55 PM 12:56 AM  Liquid Dead (67 mg/kg) culture:growth 19 8:00 AM meropenem + 9:00 AM 5:00 PM 7:01 PM 1:01 AM 9:00 AM5:00 PM 1:01 AM Plated: no Alive 6/6 piperacillin colonies (67 mg/kg ofeach) 20 8:01 AM meropenem + 9:01 AM 5:01 PM 7:02 PM 1:02 AM 9:01 AM5:01 PM 1:02 AM Liquid Alive piperacillin culture: no (67 mg/kg of each)growth 21 8:02 AM meropenem + 9:02 AM 5:02 PM 7:03 PM 1:03 AM 9:02 AM5:02 PM 1:03 AM Liquid Alive piperacillin culture: (67 mg/kg of each)growth 22 8:03 AM meropenem + 9:03 AM 5:03 PM 7:04 PM 1:04 AM 9:03 AM5:03 PM 1:04 AM Plated: no Alive piperacillin colonies (67 mg/kg ofeach) 23 8:04 AM meropenem + 9:04 AM 5:04 PM 7:05 PM 1:05 AM 9:04 AM5:04 PM 1:05 AM Plated: no Alive piperacillin colonies (67 mg/kg ofeach) 24 8:05 AM meropenem + 9:05 AM 5:05 PM 7:06 PM 1:06 AM 9:05 AM5:05 PM 1:06 AM Liquid Alive piperacillin culture: no (67 mg/kg of each)growth 25 8:10 AM meropenem + 9:10 AM 5:10 PM 7:11 PM 1:11 AM 9:10 AM5:10 PM 1:11 AM Liquid Alive 5/6 tazobactam culture: (67 mg/kg of each)growth 26 8:11 AM meropenem + 9:11 AM 5:11 PM 7:12 PM 1:12 AM 9:11 AM5:11 PM 1:12 AM Plated: 1 Dead tazobactam colony (67 mg/kg of each) 278:12 AM meropenem + 9:12 AM 5:12 PM 7:13 PM 1:13 AM 9:12 AM 5:12 PM 1:13AM Plated: no Alive tazobactam colonies (67 mg/kg of each) 28 8:13 AMmeropenem + 9:13 AM 5:13 PM 7:14 PM 1:14 AM 9:13 AM 5:13 PM 1:14 AMPlated: 1 Alive tazobactam colony (67 mg/kg of each) 29 8:14 AMmeropenem + 9:14 AM 5:14 PM 7:15 PM 1:15 AM 9:14 AM 5:14 PM 1:15 AM Noblood Alive tazobactam obtained (67 mg/kg of each) 30 8:15 AMmeropenem + 9:15 AM 5:15 PM 7:16 PM 1:16 AM 9:15 AM 5:15 PM 1:16 AMLiquid Alive tazobactam culture: (67 mg/kg of each) growth 31 8:20 AMpiperacillin + 9:20 AM 5:20 PM 7:21 PM 1:21 AM 9:20 AM 5:20 PM 1:21 AMPlated: 9 Dead 4/6 tazobactam colonies (67 mg/kg of each) 32 8:21 AMpiperacillin + 9:21 AM 5:21 PM 7:22 PM 1:22 AM 9:21 AM 5:21 PM 1:22 AMPlated: 1 Alive tazobactam colony (67 mg/kg of each) 33 8:22 AMpiperacillin + 9:22 AM 5:22 PM 7:23 PM 1:23 AM 9:22 AM 5:22 PM 1:23 AMPlated: 1 Alive tazobactam colony (67 mg/kg of each) 34 8:23 AMpiperacillin + 9:23 AM 5:23 PM 7:24 PM 1:24 AM 9:23 AM 5:23 PM 1:24 AMLiquid Alive tazobactam culture: no (67 mg/kg of each) growth 35 8:24 AMpiperacillin + 9:24 AM 5:24 PM 7:25 PM 1:25 AM 9:24 AM 5:24 PM 1:25 AMLiquid Alive tazobactam culture: no (67 mg/kg of each) growth 36 8:25 AMpiperacillin + 9:25 AM 5:25 PM 7:26 PM 1:26 AM 9:25 AM 5:25 PM 1:26 AMLiquid Dead tazobactam culture: (67 mg/kg of each) growth 37 8:30 AMdrug 1 + 2 + 3 9:30 AM 5:30 PM 7:31 PM 1:31 AM 9:30 AM 5:30 PM 1:31 AMLiquid Alive 6/6 (67 mg/kg of each) culture: no growth 38 8:31 AM drug1 + 2 + 3 9:31 AM 5:31 PM 7:32 PM 1:32 AM 9:31 AM 5:31 PM 1:32 AM LiquidAlive (67 mg/kg of each) culture: no growth 39 8:32 AM drug 1 + 2 + 39:32 AM 5:32 PM 7:33 PM 1:33 AM 9:32 AM 5:32 PM 1:33 AM Plated: no Alive(67 mg/kg of each) colonies 40 8:33 AM drug 1 + 2 + 3 9:33 AM 5:33 PM7:34 PM 1:34 AM 9:33 AM 5:33 PM 1:34 AM Plated: no Alive (67 mg/kg ofeach) colonies 41 8:34 AM drug 1 + 2 + 3 9:34 AM 5:34 PM 7:35 PM 1:35 AM9:34 AM 5:34 PM 1:35 AM Liquid Alive (67 mg/kg of each) culture: nogrowth 42 8:35 AM drug 1 + 2 + 3 9:35 AM 5:35 PM 7:36 PM 1:36 AM 9:35 AM5:35 PM 1:36 AM Plated: no Alive (67 mg/kg of each) colonies 43 8:40 AMpositive control − 9:40 AM 5:40 PM 7:41 PM 1:41 AM 9:40 AM 5:40 PM 1:41AM Liquid Alive 6/6 linezolid (30 mg/kg) culture: no growth 44 8:41 AMpositive control − 9:41 AM 5:41 PM 7:42 PM 1:42 AM 9:41 AM 5:41 PM 1:42AM Plated: no Alive linezolid (30 mg/kg) colonies 45 8:42 AM positivecontrol − 9:42 AM 5:42 PM 7:43 PM 1:43 AM 9:42 AM 5:42 PM 1:43 AM LiquidAlive linezolid (30 mg/kg) culture: no growth 46 8:43 AM positivecontrol − 9:43 AM 5:43 PM 7:44 PM 1:44 AM 9:43 AM 5:43 PM 1:44 AM LiquidAlive linezolid (30 mg/kg) culture: no growth 47 8:44 AM positivecontrol − 9:44 AM 5:44 PM 7:45 PM 1:45 AM 9:44 AM 5:44 PM 1:45 AMPlated: no Alive linezolid (30 mg/kg) colonies 48 8:45 AM positivecontrol − 9:45 AM 5:45 PM 7:46 PM 1:46 AM 9:45 AM 5:45 PM 1:46 AMPlated: no Alive linezolid (30 mg/kg) colonies 49 8:50 AM vehicle (30%9:50 AM 5:50 PM 7:51 PM 1:51 AM 9:50 AM 5:50 PM 1:51 AM Plated: Dead 0/6DMSO, 30% lawn of propylene glycol, bacteria 40% water) 50 8:51 AMvehicle (30% 9:51 AM 5:51 PM 7:52 PM 1:52 AM 9:51 AM 5:51 PM 1:52 AMLiquid Dead DMSO, 30% culture: propylene glycol, growth 40% water) 518:52 AM vehicle (30% 9:52 AM 5:52 PM 7:53 PM 1:53 AM 9:52 AM 5:52 PM1:53 AM Liquid Dead DMSO, 30% culture: propylene glycol, growth 40%water) 52 8:53 AM vehicle (30% 9:53 AM 5:53 PM 7:54 PM 1:54 AM 9:53 AM5:53 PM 1:54 AM Liquid Dead DMSO, 30% culture: propylene glycol, growth40% water) 53 8:54 AM vehicle (30% 9:54 AM 5:54 PM 7:55 PM 1:55 AM 9:54AM 5:54 PM 1:55 AM Plated: Dead DMSO, 30% lawn of propylene glycol,bacteria 40% water) 54 8:55 AM vehicle (30% 9:55 AM 5:55 PM 7:56 PM 1:56AM 9:55 AM 5:55 PM 1:56 AM Plated: Dead DMSO, 30% lawn of propyleneglycol, bacteria 40% water) * ~100 uL blood was plated for 3 samples tocheck for # of colonies * 100 uL of BHI was added to 3 tubes of bloodand mixed and 100 uL of mixture was added to 4.9 mL BHI broth 1 colonyfrom each plate with growth was added to BHI broth and grown o.n. and aglycerol stock prepared A glycerol stock was prepared from each liquidculture showing growth

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What is claimed is:
 1. A method for treating an infection caused by anantibiotic resistant bacterium in a subject, wherein the resistance isdue to a penicillin-binding protein 2a (PBP2a)-driven mechanism, themethod comprising administering to the subject an effective amount of acomposition comprising: i) at least one carbapenem or other suitableβ-lactam capable of binding the allosteric site of PBP2a; ii) at leastone β-lactamase inhibitor; and iii) at least one β-lactam that binds theopen configuration of the active site of PBP2a.
 2. The method of claim1, wherein the antibiotic resistant bacterium is from the genusStaphylococcus.
 3. The method of claim 1, wherein the antibioticresistant bacterium is selected from the group consisting of S. aureus,S. epidermidis, S. hominis, S. lugdunensis, S. xylosus, and S. fells. 4.The method of claim 1, wherein the antibiotic resistant bacterium ismethicillin-resistant Staphylococcus aureus (MRSA).
 5. The method ofclaim 1, wherein the at least one carbapenem or other suitable β-lactamcapable of binding the allosteric site of PBP2a is selected from thegroup consisting of meropenem, imipenem, tomopenem, ceftaroline andceftobiprole.
 6. The method of claim 1, wherein the at least onecarbapenem or other suitable β-lactam capable of binding the allostericsite of PBP2a is selected from the group consisting of meropenem andimipenem.
 7. The method of claim 1, wherein the at least one β-lactamaseinhibitor is selected from the group consisting of clavulanic acid(clavulanate), sulbactam, tazobactam and avibactam
 8. The method ofclaim 1, wherein the at least one β-lactamase inhibitor is selected fromthe group consisting of tazobactam and clavulanate.
 9. The method ofclaim 1, wherein the at least one β-lactam that binds the openconfiguration of the active site of PBP2a is selected from the groupconsisting of carbapenems, aminopenicillins, carboxypenicillins,ureidopenicillins, oxacillins, methicillins, and some cephalosporins.10. The method of claim 9, wherein the carbapenem is selected from thegroup consisting of meropenem, imipenem, doripenem, ertapenem,faropenem, and tebipenem.
 11. The method of claim 9, wherein theoxacillin or methicillin is selected from the group consisting ofcloxacillin, dicloxacillin, flucloxacillin, oxacillin, methicillin andnafcillin.
 12. The method of claim 9, wherein the cephalosporin isselected from the group consisting of cefepime, cefozopran, cefpirome,cefquinome, ceftaroline, and ceftobiprole.
 13. The method of claim 9,wherein the aminopenicillin is selected from the group consisting ofamoxicillin, ampicillin, pivampicillin, hetacillin, bacampicillin,metampicillin, talampicillin, and epicillin.
 14. The method of claim 9,wherein the carboxypenicillin is selected from the group consisting ofcarbenicillin, carindacillin, ticarcillin and temocillin.
 15. The methodof claim 9, wherein the ureidopenicillin is selected from the groupconsisting of azlocillin, mezlocillin and piperacillin.
 16. The methodof claim 9, wherein the at least one β-lactam that binds the openconfiguration of the active site of PBP2a is not mecillinam, cefradineand thienamycin.
 17. The method of claim 1, wherein the at least oneβ-lactam that binds the open configuration of the active site of PBP2ais selected from the group consisting of piperacillin and amoxicillin.18. The method of claim 1, wherein the carbapenem is meropenem, theβ-lactamase inhibitor is tazobactam and the β-lactam that binds the openconfiguration of the active site of PBP2a is piperacillin.
 19. Themethod of claim 1, wherein the carbapenem is imipenem, the β-lactamaseinhibitor is clavulanate and the β-lactam that binds the openconfiguration of the active site of PBP2a is piperacillin.
 20. Themethod of claim 1, wherein the carbapenem is meropenem, the β-lactamaseinhibitor is tazobactam, the β-lactam that binds the open configurationof the active site of PBP2a is amoxicillin and wherein the ratio of (i),(ii) and (iii) is 1:1:1.